Adenosine A2A Receptors in Substance Use Disorders: A Focus on Cocaine. Wydra K, Gawliński K, Frankowska M, Borroto-Escuela DO, Fuxe K, et al. Cells. 2020 Jun1;9(6):E1372. DOI: 10.3390/cells9061372.
Several psychoactive drugs can evoke substance use disorders (SUD) in humans and animals, and these include psychostimulants, opioids, cannabinoids (CB), nicotine, and alcohol. The etiology, mechanistic processes, and the therapeutic options to deal with SUD are not well understood. The common feature of all abused drugs is that they increase dopamine (DA) neurotransmission within the mesocorticolimbic circuitry of the brain followed by the activation of DA receptors. D2 receptors were proposed as important molecular targets for SUD. The findings showed that D2 receptors formed heteromeric complexes with other GPCRs, which forced the addiction research area in new directions. In this review, we updated the view on the brain D2 receptor complexes with adenosine (A)2A receptors (A2AR) and discussed the role of A2AR in different aspects of addiction phenotypes in laboratory animal procedures that permit the highly complex syndrome of human drug addiction. We presented the current knowledge on the neurochemical in vivo and ex vivo mechanisms related to cocaine use disorder (CUD) and discussed future research directions for A2AR heteromeric complexes in SUD.
APOE ε4 and the Influence of Sex, Age, Vascular Risk Factors, and Ethnicity on Cognitive Decline. Makkar SR, Lipnicki DM, Crawford JD, Kochan NA, Castro-Acosta E, Lima-Costa MF, et al. J Gerontol A Bio Sci Med Sci. 2020 May 12;glaa116.
DOI: 10.1093/gerona/glaa116. Online ahead of print.
We aimed to examine the relationship between APOE*4 carriage on cognitive decline, and whether these associations were moderated by sex, baseline age, ethnicity, and vascular risk factors. Participants were 19,225 individuals aged 54-103 years from 15 longitudinal cohort studies with a mean follow up duration ranging between 1.2 and 10.7 years. Two-step individual participant data (IPD) meta-analysis was used to pool results of study-wise analyses predicting memory and general cognitive decline from carriage of one or two APOE*4 alleles, and moderation of these associations by age, sex, vascular risk factors and ethnicity. Separate pooled estimates were calculated in both men and women who were younger (i.e., 62 years) and older (i.e., 80 years) at baseline. Results showed that APOE*4 carriage was related to faster general cognitive decline in women, and faster memory decline in men. A stronger dose-dependent effect was observed in older men, with faster general cognitive and memory decline in those carrying two versus one APOE*4 allele. Vascular risk factors were related to an increased effect of APOE*4 on memory decline in younger women, but a weaker effect of APOE*4 on general cognitive decline in older men. The relationship between APOE*4 carriage and memory decline was larger in older-aged Asians than Whites. In sum, APOE*4 is related to cognitive decline in men and women, although these effects are enhanced by age and carriage of two APOE*4 alleles in men, a higher numbers of vascular risk factors during the early stages of late adulthood in women, and Asian ethnicity.
Association of Childhood Smoking and Adult Mortality: Prospective Study of 120 000 Cuban Adults. Thomson B, Armas Rojas N, Lacey B, Burret JA, Varona-Pérez P, Calderón Martínez M, et al. Lancet Glob Health. 2020 Jun;8(6):e850–7. DOI: 10.1016/S2214-109X(20)30221-7.
Background The average age at which people start smoking has been decreasing in many countries, but insufficient evidence exists on the adult hazards of having started smoking in childhood and, especially, in early childhood. We aimed to investigate the association between smoking habits (focusing on the age when smokers started) and cause-specific premature mortality in a cohort of adults in Cuba. Methods For this prospective study, adults were recruited from five provinces in Cuba. Participants were interviewed (data collected included socioeconomic status, medical history, alcohol consumption, and smoking habits) and had their height, weight, and blood pressure measured. Participants were followed up until Jan 1, 2017 for cause-specific mortality; a subset was resurveyed in 2006-08. We used Cox regression to calculate adjusted rate ratios (RRs) for mortality at ages 30-69 years, comparing never-smokers with current smokers by age they started smoking and number of cigarettes smoked per day and with ex-smokers by the age at which they had quit. Findings Between Jan 1, 1996, and Nov 24, 2002, 146 556 adults were recruited into the study, of whom 118 840 participants aged 30-69 years at recruitment contributed to the main analyses. 27 264 (52%) of 52 524 men and 19 313 (29%) of 66 316 women were current smokers. Most participants reported smoking cigarettes; few smoked only cigars. About a third of current cigarette smokers had started before age 15 years. Compared with never-smokers, the all-cause mortality RR was highest in participants who had started smoking at ages 5-9 years (RR 2·51, 95% CI 2·21-2·85), followed by ages 10-14 years (1·83, 1·72-1·95), 15-19 years (1·56, 1·46-1·65), and ages 20 years or older (1·50, 1·39-1·62). Smoking accounted for a quarter of all premature deaths in this population, but quitting before about age 40 years avoided almost all of the excess mortality due to smoking. Interpretation In this cohort of adults in Cuba, starting to smoke in childhood was common and quitting was not. Starting in childhood approximately doubled the rate of premature death (ie, before age 70 years). If this 2-fold mortality RR continues into old age, about half of participants who start smoking before age 15 years and do not stop will eventually die of complications from their habit. The greatest risks were found among adults who began smoking before age 10 years.
Cardiac Vagal Imbalance to the Isometric Sustained Weight Test in Adolescents With Emotional Eating Behavior. González-Vázquez VE, Pedraza-Rodríguez EM, Carrazana-Escalona R, Moreno-Padilla M, Muñoz-Bustos GA, Sánchez-Hechavarría ME. Physiol Behav. 202 Jun 2;223:112994.
DOI: 10.1016/j.physbeh.2020.112994. Online ahead of print.
Purpose To assess the relationship between emotional eating behavior and heart rate variability in Spanish adolescents during an isometric exercise test. Methods Participants included 52 adolescents aged between 13 and 18 years old. Heart rate was continuously recorded at rest (2 minutes) and during the sustained weight test (2 minutes). Linear and nonlinear methods of heart rate variability were assessed and related to the emotional eating behavior divided in two clusters. Results Statistically significant differences were observed in linear and non-linear parameters of heart rate variability comparing rest and sustained weight test. An increase in the value of emotional eating in overweight adolescents was founded. During the sustained weight test, there were differences between the two emotional eating clusters regarding the variables peak high frequency power, normalized low frequency power, normalized high frequency power, low frequency/high frequency ratio, and sample entropy. A positive correlation between the emotional eating behavior and the peak high frequency power was observed, though the prediction capacity of the high frequency waves is low it is observed that there is a good fit to the regression line. Conclusion Results of this study shows that there was a relationship between vagal tone and emotional eating behavior in adolescents during an isometric exercise, with excessive parasympathetic predominance and sympathetic withdrawal during a physical effort.
CIGB-300 Anticancer Peptide Regulates the Protein Kinase CK2-dependent Phosphoproteome. Perera Y, Ramos Y, Padrón G, Caballero E, Guirola O, Caligiuri LG, et al. Mol Cell Biochem. 2020 May 13.
DOI: 10.1007/s11010-020-03747-1. Online ahead of print.
Casein-kinase CK2 is a Ser/Thr protein kinase that fosters cell survival and proliferation of malignant cells. The CK2 holoenzyme, formed by the association of two catalytic alpha/alpha’ (CK2α/CK2α’) and two regulatory beta subunits (CK2β), phosphorylates diverse intracellular proteins partaking in key cellular processes. A handful of such CK2 substrates have been identified as targets for the substrate-binding anticancer peptide CIGB-300. However, since CK2β also contains a CK2 phosphorylation consensus motif, this peptide may also directly impinge on CK2 enzymatic activity, thus globally modifying the CK2-dependent phosphoproteome. To address such a possibility, firstly, we evaluated the potential interaction of CIGB-300 with CK2 subunits, both in cell-free assays and cellular lysates, as well as its effect on CK2 enzymatic activity. Then, we performed a phosphoproteomic survey focusing on early inhibitory events triggered by CIGB-300 and identified those CK2 substrates significantly inhibited along with disturbed cellular processes. Altogether, we provided here the first evidence for a direct impairment of CK2 enzymatic activity by CIGB-300. Of note, both CK2-mediated inhibitory mechanisms of this anticancer peptide (i.e., substrate- and enzyme-binding mechanism) may run in parallel in tumor cells and help to explain the different anti-neoplastic effects exerted by CIGB-300 in preclinical cancer models.
CIGB-552, a Promising Candidate to Cancer Therapy. Oliva Arguelles B, Riera-Romo M, Guerra Vallespi M. Br J Pharmacol. 2020 May 20.
DOI: 10.1111/bph.15132. Online ahead of print.
Peptide-based cancer therapy has been of great interest due to the unique advantages of peptides, such as the low molecular weight, the ability to specifically target tumor cells, easy availability and low toxicity in normal tissues. Therefore, identify and synthesize novel peptides could provide a promising choice to cancer patients. The antitumor second generation peptide CIGB-552 has been developed as a candidate to cancer therapy. Proteomic and genomic studies have identified the intracellular protein COMMD1 as the specific target of CIGB-552. This peptide penetrates inside tumor cells to induce the proteasomal degradation of RelA, causing the termination of NF-kB signaling. The antitumor activity of CIGB-552 has been validated in vitro in different human cancer cell lines, as well as in vivo in syngeneic and xenograft tumor mouse models and in cancer-bearing dogs. The aim of this review is to present and discuss the experimental data about CIGB-552, its mechanism of action and its therapeutic potential in human chronic diseases. This peptide is already in phase I of clinical trials as antineoplastic drug, but also has possible application to other inflammatory and metabolic conditions.
Duration in Male Spinocerebellar Ataxia Type 2 Patients. Almaguer-Mederos LE. Aguilera-Rodríguez R, Almaguer-Gotay D, Hechavarría-Berzaga K, Álvarez-Sosa A, Chapman-Rodríguez Y, et al. Cerebellum. 2020 May 21.
DOI: 10.1007/s12311-020-01134-6. Online ahead of print.
Spinocerebellar ataxia type 2 (SCA2) is a progressive neurodegenerative disorder due to an unstable expansion of a CAG repeat in the ATXN2 gene. Despite clinical and experimental evidence indicating the relevance of the gonadotropic axis to the prognosis and therapeutics for several late-onset neurodegenerative disorders, its functioning and association with disease severity have not been previously explored in SCA2. To assess serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), and their clinical relevance in SCA2 patients. A case-control study involving 94 Cuban SCA2 patients and 101 gender- and age-matched healthy controls was conducted. Testosterone, LH, and FSH serum levels were determined by radioimmunoassay or immunoradiometric assay systems. Clinical outcomes included age at onset, disease duration, Scale for the Assessment and Rating of Ataxia (SARA) score, and progression rate. Univariate general linear models were generated. Testosterone, LH, and FSH serum levels were significantly reduced in male SCA2 patients relative to control individuals. On average, there was a 35% reduction in testosterone levels in male patients versus male control individuals. Testosterone levels were associated with disease duration (r = 0.383; p = 0.025) and age at onset (r = 0.414; p = 0.011) in male SCA2 patients, but no association was observed between testosterone and CAG expansion size, SARA score, or progression rate. Testosterone levels might be a biomarker of disease progression in male SCA2 patients. Further studies are needed to explore the effects of low testosterone levels on non-motor symptoms, and to assess the potential of testosterone replacement therapy in male SCA2 patients.
Efficacy of Direct Current Generated by Multiple-Electrode Arrays on F3II Mammary Carcinoma: Experiment and Mathematical Modeling. Villar Goris NA, García Rodríguez JL, Morales González M, Olivares Borges B, Fyentes Morales D, Maine Calzado E, et al. J Transl Med. 2020 May 7;18(1):190.
DOI: 10.1186/s12967-020-02352-6.
Background The modified Gompertz equation has been proposed to fit experimental data for direct current treated tumors when multiple-straight needle electrodes are individually inserted into the base perpendicular to the tumor long axis. The aim of this work is to evaluate the efficacy of direct current generated by multiple-electrode arrays on F3II mammary carcinoma that grow in the male and female BALB/c/Cenp mice, when multiple-straight needle electrodes and multiple-pairs of electrodes are inserted in the tumor. Methods A longitudinal and retrospective preclinical study was carried out. Male and female BALB/c/Cenp mice, the modified Gompertz equation, intensities (2, 6 and 10 mA) and exposure times (10 and 20 min) of direct current, and three geometries of multiple-electrodes (one formed by collinear electrodes and two by pair-electrodes) were used. Tumor volume and mice weight were measured. In addition, the mean tumor doubling time, tumor regression percentage, tumor growth delay, direct current overall effectiveness and mice survival were calculated. Results The greatest growth retardation, mean doubling time, regression percentage and growth delay of the primary F3II mammary carcinoma in male and female mice were observed when the geometry of multiple-pairs of electrodes was arranged in the tumor at 45, 135, 225 and 325o and the longest exposure time. In addition, highest direct current overall effectiveness (above 66%) was observed for this EChT scheme. Conclusions It is concluded that electrochemical therapy may be potentially addressed to highly aggressive and metastic primary F3II murine mammary carcinoma and the modified Gompertz equation may be used to fit data of this direct current treated carcinoma. Additionally, electrochemical therapy effectiveness depends on the exposure time, geometry of multiple-electrodes and ratio between the direct current intensity applied and the polarization current induced in the tumor.
EMG Rectification Is Detrimental for Identifying Abnormalities in Corticomuscular and Intermuscular Coherence in Spinocerebellar Ataxia Type 2. Ruiz-González Y, Velázquez-Pérez L, Rodríguez-Labrada, Torres-Vega R, Ziemann U. Cerebellum. 2020 Jun 4.
DOI: 10.1007/s12311-020-01149-z. Online ahead of print.
Corticomuscular and intermuscular coherence (CMC, IMC) reflect connectivity between neuronal activity in the motor cortex measured by electroencephalography (EEG) and muscular activity measured by electromyography (EMG), or between activity in different muscles, respectively. There is an ongoing debate on the appropriateness of EMG rectification prior to coherence estimation. This work examines the effects of EMG rectification in CMC and IMC estimation in 20 spinocerebellar ataxia type 2 (SCA2) patients, 16 prodromal SCA2 gene mutation carriers, and 26 healthy controls during a repetitive upper or lower limb motor task. Coherence estimations were performed using the non-rectified raw EMG signal vs. the rectified EMG signal. EMG rectification decreases the level of significance of lower beta-frequency band CMC and IMC values in SCA2 patients and prodromal SCA2 mutation carriers vs. healthy controls, and also results in overall lower coherence values. EMG rectification is detrimental for beta-frequency band CMC and IMC estimation. One likely reason for this effect is distortion of coherence estimation in high-frequency signals, where the level of amplitude cancelation is high.
Evidence for the Existence of A2AR-TrkB Heteroreceptor Complexes in the Dorsal Hippocampus of the Rat Brain: Potential Implications of A2AR and TrkB Interplay Upon Ageing. Di Palma M, Sartini S, Lattanzi D, Cuppini R, Pita-Rodríguez M, Díaz-Carmenate Y, et al. Mech Ageing Dev. 2020 Jun 18;190:111289. DOI: 10.1016/j.mad.2020.111289. Online ahead of print.
Adenosine A2A receptors (A2AR) are crucial in facilitating the BDNF action on synaptic transmission in the rat hippocampus primarily upon ageing. Furthermore, it has been suggested that A2AR-Tropomyosin related kinase B receptor (TrkB) crosstalk has a pivotal role in adenosine A2AR-mediated modulation of the BDNF action on hippocampal plasticity. Considering the impact of the above receptors interplay on what concerns BDNF-induced enhancement of synaptic transmission, gaining a better insight into the mechanisms behind this powerful crosstalk becomes of primary interest. Using in situ proximity ligation assay (PLA), the existence of a direct physical interaction between adenosine A2AR and TrkB is demonstrated. The A2AR-TrkB heteroreceptor complexes show a heterogeneous distribution within the rat dorsal hippocampus. High densities of the heteroreceptor complexes were observed in the pyramidal cell layers of CA1-CA3 regions and in the polymorphic layer of the dentate gyrus (DG). The stratum radiatum of the CA1-3 regions showed positive PLA signal in contrast to the oriens region. The molecular and granular layers of the DG also lacked significant densities of PLA positive heteroreceptor complexes, but subgranular zone showed some PLA positive cells. Their allosteric receptor-receptor interactions may significantly modulate BDNF signaling impacting on hippocampal plasticity which is impaired upon ageing.
Features of Speech and Swallowing Dysfunction in Pre-Ataxic Spinocerebellar Ataxia Type 2. Vogel AP, Magee M, Torres-Vega R, Medrano-Montero J, Cyngler MP, Kruse M, et al. Neurology. 2020 Jun 11;
DOI: 10.1212/WNL.0000000000009776. Online ahead of print.
Objective To determine whether objective and quantitative assessment of dysarthria and dysphagia in spinocerebellar ataxia type 2 (SCA2), specifically at pre-ataxic and early disease phases, can act as sensitive disease markers. Methods Forty-six individuals (16 with pre-ataxic SCA2, 14 with early-stage ataxic SCA2, and 16 healthy controls) were recruited in Holguin, Cuba. All participants underwent a comprehensive battery of assessments including objective acoustic analysis, clinician-derived ratings of speech function and swallowing, and quality of life assessments of swallowing. Results Reduced speech agility manifest at the pre-ataxic stage was observed during diadochokinetic tasks, with the magnitude of speech deficit augmented in the early ataxic stage. Speech rate was slower in early-stage ataxic SCA2 compared with pre-ataxic SCA2 and healthy controls. Reduced speech agility and speech rate correlated with disease severity and time to ataxia onset, verifying that speech deficits occur prior to ataxia onset and increase in severity as the disease progresses. Whereas dysphagia was observed in both pre-ataxic and ataxic SCA2, it was not associated with swallowing-related quality of life, disease severity, or time to ataxia onset. Conclusions Speech and swallowing deficits appear sensitive to disease progression in early-stage SCA2, with syllabic rate a viable marker. Findings provide insight into mechanisms of disease progression in early-stage SCA2, signaling an opportunity for stratifying early-stage SCA2 and identifying salient markers of disease onset as well as outcome measures in future early-stage therapeutic studies.
Formate Induces a Metabolic Switch in Nucleotide and Energy Metabolism. Oizel K, Tait-Mulder J, Fernández-de-Cossio-Díaz J, Pietzke M, Brunton H, Lilla S, et al. Cell Death Dis 2020 May 4;11(5):310.
DOI: 10.1038/s41419-020-2523-z.
Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP-dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following and intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism.
Fronto-Orbital Advance in a Patient With Roberts Syndrome. Jiménez Álvarez JA, Chacón LA, Duque Montealegre JA, Valdés Reyes JM, Pérez Cataño C. J Craniofac Surg. 2020 Jun 15.
DOI: 10.1097/SCS.0000000000006577. Online ahead of print.
Roberts Syndrome is an extremely rare syndrome reporting about 150 cases in the literature, with a very low survival rate. The authors present a case of a female patient with Roberts Syndrome who also had a coronal craniosynostosis. The aim of this case report is to present a case of a patient with Roberts Syndrome with a brachycephaly that required management of fronto-orbital advancement. In conclusion Roberts Syndrome is a rare disease, which can have different skeletal variations. This syndrome can manifest itself with craniosynostosis, with the requirement of a comprehensive management to correct it and avoid compression of the brain with endocranial hypertension.
General Care in the Management of Severe Traumatic Brain Injury: Latin American Consensus. Godoy DA, Videtta W, Santa Cruz R, Silva X, Aguilera-Rodríguez S, Carreño-Rodríguez JN, et al. Med Intensiva. 2020 May 3;S0210-5691(20)30060-7.
DOI: 10.1016/j.medin.2020.01.014. Online ahead of print.
Severe traumatic brain injury (sTBI) remains prevalent in the young adult population. Indeed, far from descending, the incidence of sTBI remains high. One of the key bases of treatment is to avoid, detect and correct secondary injuries of systemic origin, which aggravate the primary lesion. Much of this can be achieved by maintaining an adequate physiological microenvironment allowing recovery of the damaged brain tissue. General care measures are nonspecific actions designed to meet that objective. The available guidelines on the management of sTBI have not included the topics contemplated in this consensus. In this regard, a group of members of the Latin American Brain Injury Consortium (LABIC), involved in the different aspects of the acute management of sTBI (neurosurgeons, intensivists, anesthesiologists, neurologists, nurses and physiotherapists) were gathered. An exhaustive literature search was made of selected topics in the LILACS, PubMed, Embase, Scopus, Cochrane Controlled Register of Trials and Web of Science databases. To establish recommendations or suggestions with their respective strength or weakness, the GRADE methodology (Grading of Recommendations, Assessment, Development and Evaluation) was applied. Additionally, certain recommendations (included in complementary material) were not assessed by GRADE, because they constitute a set of therapeutic actions of effective compliance, in which it was not possible to apply the said methodology. Thirty-two recommendations were established, 16 strong and 16 weak, with their respective levels of evidence. This consensus attempts to standardize and establish basic general care measures in this particular patient population.
Genotyping of Treponema Pallidum in Cuba (2018-2019): Increased Circulation of Recombinant Genotype and No New Treponema Pallidum Subsp. Endemicum Infection Among Syphilis Patients. Noda AA, Rodríguez I, Šmajs D. Sex Transm Dis. 2020 Jun 8.
DOI: 10.1097/OLQ.0000000000001217. Online ahead of print.
Objectives To determine the allelic profiles of T. pallidum in patients confirmed with syphilis in Cuba (2018-2019) and to explore mutations leading to macrolide and tetracycline resistance. Methods Multi-locus sequence typing and PCR of rrn loci (23S and 16S rDNA), followed by Sanger sequencing, was used to define the allelic profile of TPA and resistance mutations, respectively. Results Allelic profile 1.3.1 and the recombinant profile were identified, with 15.7.3 having an increased frequency. We did not detect the presence of the T. pallidum subsp. endemicum among syphilis patients, as in previous reports. A high frequency of macrolide-resistant strains and the absence of mutations potentially causing tetracycline resistance was found. Conclusions Understanding the current status of treponemal infection in Cuban patients provides insights into the syphilis epidemiology.
Learning About Mental Healthcare in Today’s Cuba: An Interview With the President of the Cuban Society of Psychology. Linz SJ, Ruiz AL. Perspect Psychiatr Care. 2020 Jun 7.
DOI: 10.1111/ppc.12548. Online ahead of print.
Purpose Little is known about Cuba’s mental healthcare system. We present background information and an interview with the President of the Cuban Society of Psychology to learn about current mental healthcare in today’s Cuba. Conclusions Mental and medical healthcare are free and fully integrated. Early diagnosis and intervention is standard as each patient is known by their community doctor/nurse team from infancy through old age and by yearly home visits. Practice implications Free and integrated medical and mental healthcare facilitates early detection and intervention. Individuals in Cuba are assisted in maintaining job and schooling during treatment. Therapy is multimodal and eclectic.
Improved Production of the Recombinant Phospholipase A1 From Polybia paulista Wasp Venom Expressed in Bacterial Cells for Use in Routine Diagnostics. Pérez-Rivero A, Musacchio-Lasa A, Romani Fernandes LG, Dos Santos-Pinto JRA, Grego Esteves F, Luiz Bazon M, et al. 3 Biotech. 2020 May;10(5):217.
DOI: 10.1007/s13205-020-02202-8. Epub 2020 Apr 27.
Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate-phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy.
Loss of Ventricular Pre-excitation During Non-invasive Testing Does Not Exclude High-Risk Accessory. Escudero CA, Ceresnak SR, Collins KK, Pass RH, Aziz PF, Blaufox AD, et al. Heart Rhythm. 2020 Jun 1. Online ahead of print.
Background Abrupt loss of ventricular preexcitation on noninvasive evaluation, or nonpersistent preexcitation, in Wolff-Parkinson-White syndrome (WPW) is thought to indicate a low risk of life-threatening events. Objective The purpose of this study was to compare accessory pathway (AP) characteristics and occurrences of sudden cardiac arrest (SCA) and rapidly conducted preexcited atrial fibrillation (RC-AF) in patients with nonpersistent and persistent preexcitation. Methods Patients 21 years or younger with WPW and invasive electrophysiology study (EPS) data, SCA, or RC-AF were identified from multicenter databases. Nonpersistent preexcitation was defined as absence/sudden loss of preexcitation on electrocardiography, Holter monitoring, or exercise stress test. RC-AF was defined as clinical preexcited atrial fibrillation with shortest preexcited R-R interval (SPERRI) ≤ 250 ms. AP effective refractory period (APERP), SPERRI at EPS, and shortest preexcited paced cycle length (SPPCL) were collected. High-risk APs were defined as APERP, SPERRI, or SPPCL ≤ 250 ms. Results Of 1589 patients, 244 (15%) had nonpersistent preexcitation and 1345 (85%) had persistent preexcitation. There were no differences in sex (58% male vs 60% male; P=.49) or age (13.3±3.6 years vs 13.1±3.9 years; P=.43) between groups. Although APERP (344±76 ms vs 312±61 ms; P<.001) and SPPCL (394±123 ms vs 317±82 ms; P<.001) were longer in nonpersistent vs persistent preexcitation, there was no difference in SPERRI at EPS (331±71 ms vs 316±73 ms; P=.15). Nonpersistent preexcitation was associated with fewer high-risk APs (13% vs 23%; P<.001) than persistent preexcitation. Of 61 patients with SCA or RC-AF, 6 (10%) had nonpersistent preexcitation (3 SCA, 3 RC-AF). Conclusion Nonpersistent preexcitation was associated with fewer high-risk APs, though it did not exclude the risk of SCA or RC-AF in children with WPW.
Nanocarriers as Potential Drug Delivery Candidates for Overcoming the Blood-Brain Barrier: Challenges and Possibilities. Ahlawa J, Barroso GG, Asil SM, Alvarado M, Armendariz I, Bernal J, et al. ACS Omega. 2020 Jun 1;5(22):12583–95.
DOI: 10.1021/acsomega.0c01592.
The design of a drug that successfully overcomes the constraints imposed by the blood-brain barrier (BBB, which acts as a gatekeeper to the entry of substances into the brain) requires an understanding of the biological firewall. It is also of utmost importance to understand the physicochemical properties of the said drug and how it engages the BBB to avoid undesired side effects. Since fewer than 5% of the tested molecules can pass through the BBB, drug development pertaining to brain-related disorders takes inordinately long to develop. Furthermore, in most cases it is also unsuccessful for allied reasons. Several drug delivery systems (DDSs) have shown excellent potential in drug delivery across the BBB while demonstrating minimal side effects. This mini-review summarizes key features of the BBB, recapitulates recent advances in our understanding of the BBB, and highlights existing strategies for the delivery of drug to the brain parenchyma.
Partial Recovery of Vegetative State After a Massive Ischaemic Stroke in a Child With Sickle Cell Anaemia. Machado C, Rodríguez-Rojas R, LEisman G. BMJ Case Rep. 2020 May 5;13(5):e233737.
DOI: 10.1136/bcr-2019-233737.
A 15-year-old patient with sickle cell disease with recessive homozygous haemoglobin S/HbSS suffered several crises developmentally after the last of which the patient fell into coma. CT scan then revealed a large infarct of the right cerebral hemisphere. Three weeks after the event, the patient began to demonstrate spontaneous eye opening and spastic quadriparesis with no evidence of command-following, gestural or verbal communication, visual pursuit or purposeful motor behaviour. Our case was in an ‘unresponsive wakefulness syndrome’ with atrophy of lateral and frontal regions of both hemispheres, demonstrated by MRI and preservation of circulation in the posterior arterial system, documented by MR angiography. Currently observed are spontaneous eye opening, preserved visual and auditory startle reflexes, normal brainstem reflexes, and grasp, palmomental and sucking reflexes. Our case demonstrates partial recovery of awareness with significant brain lesions, reflecting preserved brain activity as an indication of the modular nature of functional networks.
Pathways: A Multicenter Study of WPW in Children. Escudero CA, Ceresnak SR, Collins KK, Pass RH, Aziz PF, Blaufox AD, et al. Heart Rhythm. 2020 Jun 1;S1547-5271(20)30533-6. DOI: 10.1016/j.hrthm.2020.05.035. Online ahead of print.
Background Abrupt loss of ventricular pre-excitation on non-invasive evaluation, or non-persistent pre-excitation, in Wolff-Parkinson-White syndrome (WPW) is thought to indicate a low risk of life-threatening events. Objective To compare accessory pathway (AP) characteristics and occurrences of sudden cardiac arrest (SCA) and rapidly conducted pre-excited atrial fibrillation (RC-AF) in patients with non-persistent and persistent pre-excitation. Methods Patients ≤21 years with WPW and invasive electrophysiology study (EPS) data, SCA, or RC-AF were identified from multicenter databases. Non-persistent pre-excitation was defined as absence/sudden loss of pre-excitation on ECG, Holter, or exercise test. RC-AF was defined as clinical pre-excited atrial fibrillation with shortest pre-excited R-R interval (SPERRI) ≤250ms. AP effective refractory period (APERP), SPERRI at EPS (EPS-SPERRI), and shortest pre-excited paced cycle length (SPPCL) were collected. High-risk APs were defined as APERP, SPERRI, or SPPCL ≤250ms. Results Of 1589 patients, 244 (15%) had non-persistent pre-excitation and 1345 (85%) had persistent pre-excitation. There were no differences in sex (58 vs 60% male, p=0.49) or age (13.3±3.6 vs 13.1±3.9 years, p=0.43) between groups. Though APERP (344±76 vs 312±61ms, p<0.001), and SPPCL (394±123 vs 317±82ms, p<0.001) were longer in non-persistent versus persistent pre-excitation, there was no difference in EPS-SPERRI (331±71 vs 316±73ms, p=0.15). Non-persistent pre-excitation was associated with fewer high-risk APs (13 vs 23%, p<0.001) than persistent pre-excitation. Of 61 patients with SCA or RC-AF, 6 (10%) had non-persistent pre-excitation (3 SCA, 3 RC-AF). Conclusion Non-persistent pre-excitation was associated with fewer high-risk APs, though it did not exclude risk of SCA or RC-AF in children with WPW.
Perspectives on Battling COVID-19 in Countries of Latin America and the Caribbean. Andrus JK, Evans-Gilbert T, Santos JI, Guzmán MG, Rosenthal PJ, Toscano C, et al. Am J Trop Med Hyg. 2020 Jun 9.
DOI: 10.4269/ajtmh.20-0571. Online ahead of print.
The first case of COVID-19 reported in Latin America occurred in São Paolo, Brazil, on February 25, 2020, in a 61-yearold man who had recently returned from the Lombardy region in Italy. The first cases in the Caribbean region were reported on March 1 in St Martin in a couple who returned from France and in the Dominican Republic in a 61-year-old man visiting from Italy.1,2 By mid-March, there was a substantial surge in cases, resulting in nearly every country in Latin America and the Caribbean (LAC) reporting COVID-19 (Table 1). These data are reported to the Pan American Health Organization (PAHO) regional office from national ministries of health and as such may have inherent weaknesses of timing, completeness, and accuracy. However, they do provide insight on trends and hotspots in the region.
Pneumocystis Jirovecii and Microsporidia: An Unusual Coinfection in HIV Patients? de Armas Y, Capó V, Bornay-Linares FJ, Del Águila C, Matos O, Calderón EJ. Med Mycol. 2020 Jun 4;myaa048.
DOI: 10.1093/mmy/myaa048. Online ahead of print.
Pneumocystis jirovecii and microsporidia species are recognized as opportunistic infectious pathogens in AIDS patients. Coinfection of both in one patient has been rarely reported. The aim of the present study was to investigate the coinfection of P. jirovecii and microsporidia in different tissues from AIDS deceased patients. Post mortem histological finding of P. jirovecii and microsporidia was demonstrated by means of the Grocott’s methenamine silver and Brown Brenn staining, respectively. Molecular technique was used for identification and characterization of both fungi. Out of the 514 autopsied cases P. jirovecii and microsporidia species were identified in 53 (10.3%) and 62 (12.1%) cases respectively. A total of five cases (0.97%) coinfected with Pneumocystis and microsporidia were recovered from all analyzed autopsies. Coinfection of Pneumocystis and microsporidia is very challenging and raises interesting issues about host-parasite relationship. The early diagnosis of both pathogens must be crucial to establish correct and early treatments, improve the patient’s evolution, reducing the risk of death.
QT Interval Greater Than 460 Ms in Multiple Electrocardiograms During Follow-Up in Patients With Brugada Syndrome: What Does It Contribute? Puga Bravo M, Castro J, Gallardo Y. Rev Port Cardiol. 2020 May 13;S0870-2551(20)30144-X.
DOI: 10.1016/j.repc.2019.07.009. Online ahead of print.
Introduction Corrected QT interval (QTc) >460 ms in the right precordial leads has been described as a predictor of malignant ventricular arrhythmias (MVA) in patients with Brugada syndrome (BrS). Objective To assess the presence of QTc>460 ms in multiple electrocardiograms (ECGs) during follow-up as a predictor of recurrence of MVA in patients with BrS. Methods The study group included 43 patients with BrS and an implantable cardioverter-defibrillator. ECGs were performed serially between June 2000 and January 2017. QT interval was measured and QTc was obtained by Bazett’s formula. The sample was divided into three groups: Group 1 (patients with no ECGs with QTc>460 ms); Group 2 (patients with only one ECG with QTc>460 ms); and Group 3 (patients with two or more ECGs with QTc>460 ms). Results The following variables were more frequently observed in Group 3: family history of sudden death (p=0.023), previous history of cardiorespiratory arrest (p=0.032), syncope (p=0.039), documented MVA (p=0.002), and proportion of ECGs with coved-type ST interval during follow-up (p=0.002). In Group 3, 67% of BrS patients had events during follow-up, as opposed to only 22% of Group 1 and 14% of Group 2 (Group 1 vs. Group 2, p=0.33015; Group 1 vs. Group 3, p=0.04295; and Group 2 vs. Group 3, p=0.04155). Conclusions QTc>460 ms in more than one ECG during follow-up increases the risk of MVA events in patients with BrS.
Radiolabeled Liposomes and Lipoproteins as Lipidic Nanoparticles for Imaging and Therapy. Aranda-Lara L, Morales-Avila E, Luna-Gutiérrez MA, Olivé-Alvarez E, Issac-Olivé K. Chem Phys Lipids. 2020 Jun 17;230:104934. DOI: 10.1016/j.chemphyslip.2020.104934.
Online ahead of print.
Radiolabeled lipidic nanoparticles, particularly liposomes and lipoproteins, are of great interest as agents for imaging and therapy, due not only to their peculiar physicochemical and biological properties, but also to their great versatility and the ability to manipulate them to obtain the desired properties. This review provides an overview of radionuclide labeling strategies for preparing diagnostic and therapeutic nanoparticles based on liposomes and lipoproteins that have been developed to date, as well as the main quality control methods and in vivo applications.
Resting EEG Effective Connectivity at the Sources in Developmental Dysphonetic Dyslexia. Differences With Non-Specific Reading Delay. Bosch-Bayard J, Girini K, Biscay RJ, Valdés-Sosa P, Evans AC, Chiarenza GA. Int J Psychophysiol . 2020 May 5;153:135–47.
DOI: 10.1016/j.ijpsycho.2020.04.021. Online ahead of print.
Previous studies conducted on subjects with dysphonetic dyslexia (DD) reported inefficient timing integration of information from various brain areas. This dysregulation has been referred as neuronal dyschronia or timing deficiency. The present study examines the effective brain connectivity in Dysphonetic Dyslexic subjects (DD) compared to a group of subjects with non-specific reading delay (NSRD). The hypothesis is that the timing defect should be reflected also in the effective connectivity and the subjects with developmental dyslexia have an altered information flow different from the group of children with non-specific reading delay. The quantitative EEG at the sources of 184 children with DD was compared with that of 43 children with NRSD. The Isolated Effective Coherence (iCoh) was calculated among 17 brain regions data driven selected. To assess statistical differences in the EEG connectivity between the two groups, a Linear Mixed Effect (LME) model was applied. Two very important areas perform as hubs in the information flow: one is the left calcarine sulcus, which is more active in the DD group. The second is the left rolandic operculum, which is more active in the NSRD group. In the DD group, the calcarine sulcus is sending information to the right postcentral gyrus, the left paracentral gyrus, the right angular gyrus and the right supplementary motor area. This flow of information occurs in almost all frequency bands, including delta and theta band. Slow connections may indicate less efficient or even pathological information flow. We consider this as a neurophysiological evidence of Boder’s model of dyslexia.
Socioeconomic Position and the Health Gradient in Cuba: Dimensions and Mechanisms. Nie P, Ding L, Sousa-Poza A, Alfonso León A, Xue H, Jia P, et al. BMC Public Health. 2020 Jun 5;20(1):866.
DOI: 10.1186/s12889-020-08980-3.
Background To throw light on the under-researched association between socioeconomic position (SEP) and health in Cuba, this study examined SEP gradients in health and their underlying mechanisms among urban Cuban adults aged 18-65. Methods By applying linear regressions to data from the 2010 National Survey on Risk Factors and Chronic Diseases, the analysis explored the SEP-health gradient along three SEP dimensions – education, occupation, and skin colour – using ten health measures: self-reported health (SRH), general and abdominal obesity, hypertension, high glucose, high cholesterol, high triglycerides, low high-density lipoprotein cholesterol, metabolic syndrome, and cumulative risk factors. Regressions also included behaviours and health-related risk perceptions (tobacco and alcohol consumption, diet, physical activity, and risk-related behaviours). It thus investigated the SEP-health gradient and its underlying mechanisms via both behaviours and health-related risk perceptions. Results Once controlling for gender, age, marital status, region and provincial dummies, the analysis detected educational gradients in SRH (estimated coefficient [95% CI]: middle-level education = 3.535 [1.329, 5.741], p < 0.01; high-level education = 5.249 [3.050, 7.448], p < 0.01) that are partially explainable by both health-affecting behaviours (tobacco and alcohol consumption, diet, physical and sedentary activity) and risk perceptions. Using objective measures of health, however, it found no SEP-health gradients other than hypertension among people identified as having Black skin color (adjusted for demographic variables, 0.060 [0.018, 0.101], p < 0.01) and high cholesterol among those identified as having Mulatto or Mestizo skin color (adjusted for demographic variables, – 0.066 [- 0.098, – 0.033], p < 0.01). Conclusions In terms of objective health measures, the study provides minimal evidence for an SEP-health gradient in Cuba, results primarily attributable to the country’s universal healthcare system – which offers full coverage and access and affordable medications – and its highly developed education system.
Testosterone Levels Are Decreased and Associated With Disease Duration in Male Spinocerebellar Ataxia Type 2 Patients. Almaguer-Mederos LE, Aguilera-Rodríguez R, Almaguer-Gotay D, Hechevarría-Barzaga K, Álvarez-Sosa A, Chapman-Rodríguez Y, et al. Cerebellum. 2020 May 21.
DOI: 10.1007/s12311-020-01134-6. Online ahead of print.
Spinocerebellar ataxia type 2 (SCA2) is a progressive neurodegenerative disorder due to an unstable expansion of a CAG repeat in the ATXN2 gene. Despite clinical and experimental evidence indicating the relevance of the gonadotropic axis to the prognosis and therapeutics for several late-onset neurodegenerative disorders, its functioning and association with disease severity have not been previously explored in SCA2. To assess serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), and their clinical relevance in SCA2 patients. A case-control study involving 94 Cuban SCA2 patients and 101 gender- and age-matched healthy controls was conducted. Testosterone, LH, and FSH serum levels were determined by radioimmunoassay or immunoradiometric assay systems. Clinical outcomes included age at onset, disease duration, Scale for the Assessment and Rating of Ataxia (SARA) score, and progression rate. Univariate general linear models were generated. Testosterone, LH, and FSH serum levels were significantly reduced in male SCA2 patients relative to control individuals. On average, there was a 35% reduction in testosterone levels in male patients versus male control individuals. Testosterone levels were associated with disease duration (r = 0.383; p = 0.025) and age at onset (r = 0.414; p = 0.011) in male SCA2 patients, but no association was observed between testosterone and CAG expansion size, SARA score, or progression rate. Testosterone levels might be a biomarker of disease progression in male SCA2 patients. Further studies are needed to explore the effects of low testosterone levels on non-motor symptoms, and to assess the potential of testosterone replacement therapy in male SCA2 patients.
Type 2 Diabetes and Metformin Use Associate With Outcomes of Patients With Non-alcoholic Steatohepatitis-related, Child-Pugh A Cirrhosis. Vilar-Gómez E, Calzadilla-Bertot L, Wai-Sung Wong V, Castellanos M, Aller-de la Fuente R, Eslam, M, et al. Clin Gastroenterol Hepatol. 2020 May 7;S1542-3565(20)30633-9. DOI:10.1016/j.cgh.2020.04.083. Online ahead of print.
Background & aims Factors that affect outcomes of patients with non-alcoholic steatohepatitis (NASH) related cirrhosis are unclear. We studied associations of type 2 diabetes, levels of hemoglobin A1c (HbA1c), and use antidiabetic medications with survival and liver-related events in patients with NASH and compensated cirrhosis. Methods We collected data from 299 patients with biopsy-proven NASH with Child-Pugh A cirrhosis from tertiary hospitals in Spain, Australia, Hong Kong, and Cuba, from April 1995 through December 2016. We obtained information on presence of type 2 diabetes, level of HbA1c, and use of antidiabetic medications. Cox proportional and competing risk models were used to estimate and compare rates of transplant-free survival, hepatic decompensation, and hepatocellular carcinoma (HCC). Results Two-hundred and twelve patients had type 2 diabetes at baseline and 8/87 patients developed diabetes during a median follow-up time of 5.1 y (range, 0.5-10.0 y). A lower proportion of patients with diabetes survived the entire follow-up period (38%) than of patients with no diabetes (81%) (adjusted hazard ratio [aHR], 4.23; 95% CI, 1.93-9.29). Higher proportions of patients with diabetes also had hepatic decompensation (51% vs 26% of patients with no diabetes; aHR, 2.03; 95% CI 1.005-4.11) and HCC (25% vs 7% of patients with no diabetes; aHR, 5.42; 95% CI 1.74-16.80). Averaged annual HbA1c levels over time were not associated with outcomes. Metformin use over time was associated with a significant reduction in risk of death or liver transplantation (aHR, 0.41; 95% CI, 0.26-0.45), hepatic decompensation (aHR, 0.80; 95% CI, 0.74-0.97), and HCC (aHR, 0.78; 95% CI, 0.69-0.96). Metformin significantly reduced risk of hepatic decompensation and HCC only in subjects with HbA1c levels above 7.0% (aHR, 0.97; 95% CI, 0.95-0.99 and aHR, 0.67; 95% CI, 0.43-0.94, respectively). Conclusions In an international cohort of patients with biopsy-proven NASH and Child-Pugh A cirrhosis, type 2 diabetes increased risk of death and liver-related outcomes, including HCC. Patients who took metformin had higher rates of survival and lower rates of decompensation and HCC.
Venturicidin A, A Membrane-active Natural Product Inhibitor of ATP Synthase Potentiates Aminoglycoside Antibiotics.Yarlagadda V, Medina R, Wright GD. Sci Rep. 2020 May 18;10(1):8134.
DOI: 10.1038/s41598-020-64756-0.
Despite the remarkable advances due to the discovery and development of antimicrobials agents, infectious diseases remain the second leading cause of death worldwide. This fact underlines the importance of developing new therapeutic strategies to address the widespread antibiotic resistance, which is the major contributing factor for clinical failures of the current therapeutics. In a screen for antibiotic adjuvants, we identified a natural product from actinomycetes, venturicidin A (VentA) that potentiates the aminoglycoside antibiotic gentamicin against multidrug-resistant clinical isolates of Staphylococcus, Enterococcus, and Pseudomonas aeruginosa. Furthermore, the combination of gentamicin and VentA was bactericidal and rapidly eradicated methicillin-resistant S. aureus (MRSA). The molecular mechanism of gentamicin potentiation activity is attributed to uncoupling of ATP synthesis by VentA from electron transport presumably by blocking the proton flow through ATP synthase, which results in an elevated concentration of extracellular protons and subsequent anticipated raise in gentamicin uptake. The disruption of the proton flux was characterized by perturbed membrane potential in MRSA. These results demonstrate that inhibition of ATP synthase along with the subsequent membrane dysregulation, as shown here with VentA, complements aminoglycoside antibiotics against MDR bacteria, and that this approach may be employed to combat bacterial resistance.
Vitamin D, Preeclampsia and Prematurity: A Systematic Review and Meta-Analysis of Observational and Interventional Studies. Aguilar-Cordero MJ, Lasserot-Cuadrado A, Mur-Villar N, León-Ríos XA, Rivero-Blanco T, Pérez-Castillo IM. Midwifery. 2020 May 6;87:102707.
DOI: 10.1016/j.midw.2020.102707. Online ahead of print.
Background Vitamin D has important functions outside of bone metabolism. Deficiency has been associated with several adverse outcomes during pregnancy such as preeclampsia and prematurity. There is an increasing body of literature on this topic with studies performed to date having produced contradictory results. Objective To synthesize the literature about vitamin D deficiency and its association with preeclampsia and prematurity in order to determine if maternal vitamin D insufficiency and/or deficiency during pregnancy is associated with the prevalence of preeclampsia and prematurity. Design A systematic review and meta-analysis of observational and interventional studies. Methods Two independent researchers reviewed the included studies according to PRISMA reporting guidelines. A protocol for this review was registered in PROSPERO with the registration number: “CRD42019136318”. Three electronic databases (PubMed, ScienceDirect and Web of Science); were searched in order to identify eligible studies. Observational and interventional studies were selected which had been published in the last 6 years, and analysed the association between maternal vitamin D concentrations during pregnancy and the development of preeclampsia and/or preterm birth. Data were extracted and presented in tables and figures. Fixed and random-effects meta-analyses were performed on the studies which provided enough sample data to calculate odds ratios. Results from both statistical methods were compared. Meta-analysis cut-off points for vitamin D insufficiency and deficiency were defined as <75nmol/L and <50nmol/L, respectively. Results Fifty-five studies met the inclusion criteria. Fixed-effects meta-analysis of the interventional studies indicated that vitamin D supplementation acts as a prevention factor for preeclampsia and prematurity. Fixed-effects meta-analysis of observational studies concluded that vitamin D insufficiency and deficiency are associated with a higher risk of developing preeclampsia. However, prematurity and vitamin D were only associated when maternal vitamin D concentrations was <75 nmol/L. Random-effects meta-analysis found no significant association between vitamin D, preeclampsia and prematurity in either observational or interventional studies. Conclusion Higher vitamin D concentrations during pregnancy could be associated with a decreased risk of preeclampsia and prematurity but statistical significance of associations depends on the study design used. Well-designed clinical trials with vitamin.
