The Role of the Human Growth Hormone Gene Family in Pregnancy

A pregnant woman´s body undergoes profound anatomical and physiological changes to accommodate the needs of the maternal-fetal unit required for a successful pregnancy. During normal pregnancy, the placenta produces a variant of human growth hormone as well as a chorionic somatomammotropin hormone. These are the placental members of the human growth hormone gene family and play a crucial role in the regulation of maternal and fetal metabolism, as well as in the growth and development of the fetus. For this reason, the scope of this chapter is to describe the differences of the biochemical and physiological roles of the hormones coded in this locus during pregnancy, the repercussions of their deficiencies, and role in some of the most prevalent pathologies during pregnancy affecting either the mother or the fetus and also to describe how pioneering sequencing of this locus allowed our laboratory to invent the first companion diagnostics test and thus contributed to the dawn of the personalized medicine era

Comparison of five commercial kits for SARS CoV 2 RT-PCR diagnosis.

Background: SARS-CoV-2 was identified as the causal agent of the COVID-19 pandemic. Its rapid spread and huge health and economic impact prompted the development of diagnostic tests to opportunely identify affected individuals as a prerequisite to quarantine them and avoid further spreading the infection. Methods: The following commercial RT-PCR kits, approved by our National Institute of Diagnostics and Epidemiological Reference of InDRE (from the Spanish name) were tested: Vircell (Granda, Spain), Light Mix Roche (Berlin, Germany), Logix Smart (Utah, USA), 1 Copy (Korea Republic), and RIDA GENE (Darmstadt, Germany). RNA was isolated either manually or automatically (QIAmp Viral RNA and SMART-32, DAAN GENE kits, respectively) from naso and oro pharyngeal swabs from suspicious individuals living in Mexico city and outskirts. Results: Since May 2020, when we received InDRE´s SARS-COV2 diagnostic approval, we have processed nearly 20,000 naso and oro-pharyngel swabs samples. The qualification of kits, as per their analytical performance, value of their controls, and convenience (mono versus multiplex) resulted in the following ranking from the most to the least convenient: 1) RIDA, 2) Vircell, 3) Roche, 4) 1 Copy, and 5) Logix Smart. Conclusions: Both, analytical performance and convenience to process quantious parallel samples in a short period of time, and particularly sensitivity, were key parameters for our laboratory to adopt either RIDA or Vircell kits. They are particularly useful in cases with low viral load, which even if asymptomatics, can be contagious for vulnerable subjects within their families, community, and at work

Production of codon optimized Polyomavirus small t antigen in Escherichia coli.

Background: Hauzen et al., went against the dogma that cancer only develops by genetic factors and postulated oncogenic human papillomavirus (HPV) as the etiological agent of cervical cancer (2008). The eleven new human polyomaviruses (HPyVs) identified have been shown to infect humans subclinically from an early age. The small t-antigen (tAg) proteins of polyomaviruses control cellular phosphorylation mechanisms leading to uncontrolled replication cycles and cancer. Mechanisms of transformation and progression to tumor cells, and cellular tropism of the new HPyVs, remain unknown. Availability of recombinant tAg (rtAg) can contribute to understanding them. Methods: tAg coding sequences derived from Merkel cell polyomavirus (MCPyV) genome were codon-optimized, synthesized, cloned in an expression vector (pGEX), and transformed into E. coli. Clones were fermented, induced for expression of the tAG cassette, and purified by IMAC (Immobilized metal affinity chromatography). Results: rtAg produced in bacteria from different expression strains demonstrated distinct expression levels. The rtAg of ~20KDa was produced at the level of 11.3 mg L-1 and folded correctly since antibody 5 anti-MCPyV recognized rtAg. Conclusions: MCPyV tAg was efficiently expressed in E. coli. The availability of rtAg from MCPyV will be useful in immunity diagnostics, structure studies, and investigation of metabolic pathways interference and cell tropism features of HPyVs infections. This especially until cell culture systems for new HPyVs are developed. Acknowledgements: Our sincere gratitude to the Dana-Farber Cancer Institute for gene and antibody samples as well as the National Council of Science and Technology (CONACYT) for the scholarship 304814 to LMRM

Chromium picolinate, biotin, and sodium bicarbonate combination as a dietary supplement in the treatment of type 2 diabetes

Background: Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia due to insulin resistance, which can lead to micro and macrovascular complications. The importance of glycemic control for prevention demands the need to promote accessible and safe treatments among others in the form of scientifically proven nutritional supplements. Previous studies have suggested that the consumption of bicarbonate-rich mineral water altered blood metabolites and gut microbiome which has beneficial effects on patients with T2DM. Likewise, chromium picolinate and biotin have shown usefulness in glycemic control. The objective of our study was to evaluate the supplementation with chromium picolinate, biotin, and sodium bicarbonate in patients with T2DM. Methods: We planned and supervised the execution of a crossover, randomized, double-blind, placebo-controlled study of patients with the diagnosis of T2DM that was conducted in Diabetes Clinics of the Endocrinology Service of the University Hospital “Dr. José E. González” of the Autonomous University of Nuevo Leon in Monterrey, Mexico from June 2011 to July 2012. Patients’ follow-ups during the study included a day-0 baseline visit and six more visits over the next six months. Efficacy of treatment was assessed by expressing changes in hemoglobin A1c (HbA1c), body mass index (BMI), and blood pressure (BP). Results: Forty-seven (62.6%) of the original 75 patients completed the trial. Regarding the baseline characteristics, 25 (53.1%) of the participants were male and the mean age was 55.23 ± 9.88. The mean HbA1c was 8.38 ± 1.08%, the mean BMI was 29.34 ± 4.64, the mean systolic BP of 143.84 ± 23.6 mm Hg, and the mean diastolic BP of 84.5 ± 12.13 mm Hg. When comparing the changes that occurred after both interventions, we observed that the HbA1c in the active ingredient group decreased (-0.15%) and in the placebo increased (+0.12%) (p=0.148). When we subdivided both groups according to their HbA1c level before the intervention and compared the participants with HbA1c ≥9, the placebo group had an increase of 0.15 ± 1.32 % and the reduction in the active ingredients was -0.68 ± 1.58 % (p=0.158). Conclusions: In our study, we observed that the supplementation with chromium picolinate, biotin, and sodium bicarbonate decreased HbA1c in 3 months compared to the placebo group in which there was an increase, but with a tendency in the statistical analyses. We believe that this could be due to two reasons: the size of our sample, due to the large percentage of participants who dropped out of the study, or because the treatment period to observe a greater difference should have been longer

Biobanking in NE Mexico for biomedical research and clinical needs

Background: The advancement of biomedicine demands tools that translate its achievements into services to both the scientific community and the pharma/biotech industry. Biobanks are powerful tools that collect, process, store, manage, and distribute biospecimens and their associated clinical/demographic data to users carrying out studies aimed at causing a real public health impact. For our laboratory to offer pharma/biotech companies support for their projects with the highest quality possible biospecimens we adopted the Best Biobanking Practices from ISBER (https://www.isber.org/). Methods: As a result of the pandemics, great effort has been dedicated to help the health ecosystem through a diagnostic service for SARS CoV-2 (by RT-PCR). Emphasis was put on proper sample collection, preanalytical characterization, and storage in ultra-low freezers. Their pre-analytical characterization included determining yield and purity by spectrophotometry using the NanoDropTM 2000 (Thermo-Fisher. Mexico City, Mexico). Results: We biobanked and supplied to internal (our Genetics laboratory) and external (validation protocols of pharma/biotech international companies) clients almost 2,000 RNA samples. Given the preanalytical qualification of the biospecimens, they performed satisfactorily for our clients’ diagnostic and innovation protocols needs. Conclusions: The biobanking services provided to both our diagnostic laboratory and to pharma/biotech companies that contracted our services delivered research materials of the highest quality. Being a private biobank recognized now nationally and internationally by public and private institutions has allowed us to participate in projects evaluating innovative diagnostics methods and devices

Correlation between Ct-values and symptoms of COVID-19 patients

Background: Currently available RT-PCR methods for the diagnosis of COVID-19 can give an estimate of the viral load. The cycle threshold value (Ct-value) of the PCR correlates inversely with the viral load; low Ct-values indicate high viral loads and vice versa. Higher viral loads have been seen to correlate with disease severity and infectivity. Therefore, we studied the correlation of the Ct-value of RT-PCR and the most common symptoms of COVID-19 individually. Methods: A prospective and descriptive study was carried out with the subjects that attended our laboratory for a COVID-19 test from September 14, 2020, to January 30, 2021. Subjects filled out a questionnaire with demographic and clinical information prior to taking the naso and oropharyngeal samples. The samples were processed by Vircell SARS-CoV-2 Real-time PCR Kit (Granada, Spain). Statistical analyses were performed using IBM SPSS software. Results: We included 657 positive subjects with complete information, with a median age of 36 (27-47) and a male predominance of 477 (72.6%). Of these, 395 (60.1%) were symptomatic and the median number of symptoms was 2 (0-5). The most predominant symptoms were headache 271 (68.6%), cough 229 (58%), and myalgias 180 (45.6%). The median Ct-value for gene N was 30 (23-36) and for gene E was 31 (23-35). In comparison between symptomatic and asymptomatic subjects, asymptomatic patients had a higher Ct-value (lower viral load) in both genes and a lower age (p Conclusions: The viral load correlates with symptoms within COVID-19, having found that higher viral loads were correlated with symptoms such as headache, cough, and fever, while lower viral loads were correlated with dyspnea, diarrhea, and alterations of smell or taste senses

Automated versus manual RNA isolation for the laboratory diagnosis of SARS-CoV-2.

Background: The rapid spread and huge health and economic impact witnessed even from the beginning of the COVID-19 pandemic prompted the development of diagnostic tests to opportunely identify individuals infected by its causing agent, the SARS CoV-2 virus. This is a prerequisite to quarantine them to avoid further spreading the infection specially to their vulnerable co-workers and family contacts. The golden standard for pathogen detection is the Polymerase Chain Reaction (PCR). To obtain an accurate result, it is important to carry out an optimal isolation of the viral RNA genome. Methods: This study aimed to compare manual (kits of Qiagen or DAAN) vs automatic (SMART-32 equipment of DAAN) RNA isolation methods. 372 samples were processed of which 200 were negative and 172 were positive of which 181 were processed manually and 191 automatically. Pre-analytical characterization of the RNA resulting from both methods included quantification of yield and qualification of purity by spectrophotometry in the Nanodrop (Thermo-Fisher, Mexico City). Results were comparatively evaluated employing the IBM SPSS Statistics software. Results: The median yield of RNA obtained by the manual method resulted higher than that rendered by the automatic method. Regarding purity (as judged by the ratios of A260/230 and A260/280) the manual method reflected better parameters than the automated one. On the other hand, when dealing with large amounts of samples, the latter was more convenient and faster. Conclusions: The manual method gives slighter better yield and purity than the automated one. However, quality wise, RNA from both methods is equally suitable for RT-PCR diagnosis of the SARS-CoV-2. The demand in the laboratory for processing large volumes in the minimal time, tips the scale to the automatic method

EL CÁNCER DE MAMA HEREDITARIO

importancia del CM como problema de saludpública se refleja en el hecho de que cerca detrescientas mil mujeres mueren por esta enfermedadcada año en el mundo(1). En los Estados Unidos deNorteamérica (EUA) el CM se incrementó de unamanera sostenida desde 1930 con un aumentopromedio de 1.2% por año(2). En ese país el CMrepresenta el 32% de todos los cánceres incidentes ycomo consecuencia de esto, 50,000 mujeres muerenanualmente. Sin embargo, estudios recientes revelanuna disminución en la mortalidad en los últimosaños(3). Aunque se piensa que el aumento en la frecuencia es debido alenvejecimiento de la población y al advenimiento de mejores métodos de diagnóstico(especialmente la mamografía). También se reconoce el papel que pueden jugarcambios en el medio ambiente y en el estilo de vida, en el incremento de la frecuenciadel CM. Por otro lado, algunos de estos mismos factores y la aparición de mejoresalternativas de tratamiento, se han postulado como responsables de la disminución enla mortalidad(4)

The chimpanzee GH locus: composition, organization, and evolution

In most mammals the growth hormone (GH) locus comprises a single gene expressed primarily in the anterior pituitary gland. However, in higher primates multiple duplications of the GH gene gave rise to a complex locus containing several genes. In man this locus comprises 5 genes including GH-N (expressed in pituitary) and four genes expressed in the placenta, but in other species the number and organization of these genes varies. The situation in chimpanzee has been unclear, with suggestions of up to seven GH-like genes. We have re-examined the GH locus in chimpanzee and have deduced the complete sequence. The locus includes five genes apparently organized in a similar fashion to those in human, with two of these genes encoding GH-like proteins, and three encoding chorionic somatomammotropins/placental lactogens (CSHs/PLs). There are notable differences between the human and chimpanzee loci with regard to the expressed proteins, gene regulation and gene conversion events. In particular, one human gene (hCSH-L) has changed substantially since the chimpanzee/human split, potentially becoming a pseudogene, while the corresponding chimpanzee gene (CSH-A1) has been conserved, giving a product almost identical to the adjacent CSH-A2. Chimpanzee appears to produce two CSHs, with potentially differing biological properties, whereas human produces a single CSH. The pattern of gene conversion in human has been quite different from that in chimpanzee. The region around the GH-N gene in chimpanzee is remarkable polymorphic, unlike the corresponding region in human. The results shed new light on the complex evolution of the GH-locus in higher primates

Rational design for the recombinant expression of the Receptor Binding Domain of SARS-CoV-2 Spike Glycoprotein

Background: COVID-19 represents a significant threat to global human health. SARS-CoV-2, the etiologic viral agent, needs to be under-covered at the structural biology level to facilitate the rational design of diagnostic tests and vaccine candidates. SARS-CoV-2 Receptor Binding Domain of Spike protein (RBD-S) acts as the key to open the gate, to enter the cells during infection. Thus, it is a stronger candidate for designing effective antigens for vaccines and diagnostics. Here, we relied on the viral DNA codifying to RBD-S to use synthetic biology for optimizing the recombinant expression of this (rRBD-S) as a proof of concept of rational designs of bioprocess for vaccine candidates and immunogens to improved rapid diagnostic tests. Methods: rRBD-S coding sequences inspired on RBD-S ectodomain from SARS-CoV-2 were designed, codon-optimized, tagged, synthesized, cloned in an expression vector (pD444-MR), and transformed into C41(DE3)pLysS E. coli strain. Expression of recombinant RBD-S was resulting in a protein purified using Ni-IMAC (Nickel Immobilized metal affinity chromatography). Results: rRBD-S produced result in a ~30KDa protein with yields of 4.618 gr L-1. Protein was recovered from the bacterial soluble fraction without refolding process. Conclusions: rRBD-S is an important tool for immunity diagnostics as Lateral-Flow-Devices, structural biology studies, and even as vaccine candidate for combating SARS-CoV-2. Notably considering the advantages of rational subunit vaccines for immune response against other vaccines technologies whose effectiveness in the long-term process has not been demonstrated yet. Acknowledgements: We thank HIDALGO´s Council of Science, Technology and Innovation (CITNOVA) for the GRANT 20201122 to LMRM