Role of Human Papilloma Virus 16/18 In Laryngeal Carcinoma with Correlation to the Expression of Cyclin D1, p53, p16 and EGFR.

Objective: To investigate the role of Human Papilloma Virus (HPV) (16/18) in relation to the molecular genetic mechanisms of Cyclin D1, p53, p16, and Epidermal growth factor receptor (EGFR) in Laryngeal Squamous Cell Carcinoma (LSSC). Methods: A cross-sectional study of 88 (Formalin-fixed Paraffin Embedded) FFPE laryngeal biopsies were done at Basic Medical Sciences Institute, Jinnah Postgraduate Centre, Karachi from 2010 to 2019 with the application of Polymerase chain reaction (PCR) for HPV 16/18and Immuno-histochemical staining for molecular genetic expression of proteins, Cyclin D1, p53, p16, and EGFR. Results: Out of 88 cases of Laryngeal Squamous Cell Carcinoma (LSSC) there was female preponderance. Mean age of the participants was found as 50.7±12.8 years. High risk HPV 16/18 was positive in 28 cases (31.8%), largely related to Grade-II and Grade-III. Immunohistochemically, Cyclin D1 (87.5%) appeared as the most important driver mutation followed by p16 (86.4%), EGFR (65.9%), and, p53 was positive in (61.4%) of cases. Conclusion: The role of high-risk HPV 16/18 is concurred in the present study strongly in correlation to p16 as a surrogate marker. Moreover, the other driver mutations of Cyclin D1, p53, and EGFR are also implicated as cumulative molecular events in tumor progression as mostly seen in higher Grades.

Profiling of inflammatory biomarkers in mild to critically ill severe acute respiratory syndrome corona virus-19 (SARS Covid-19) patients from Karachi, Pakistan.

SARS-Covid-19 infection got spread in many countries and WHO declared it as a serious global Pandemic. Pro-inflammatory cytokines storm generated by Covid-19 infection hyper-activates inflammatory response in host body, resulting in elevated release of inflammatory biomarkers. Present article describes the characteristic profile of these inflammatory and related biomarkers in a total of 48 critically ill Covid-19 patients, (Male = 38, F = 10), with mildly ill to severe, critically ill status and thus grouped accordingly. Inflammatory Biomarkers, Ferritin, ProCalcitonin, C-Reactive Protein, coagulation marker-D-Dimer, chemical analytes, Protein, Albumin, BUN, Bilirubin, Creatinine, and enzymes, Lactate Dehydrogenase, γ-Glutamyl transpeptidases, Alkaline phosphatase were routine analyzed by standard methods described earlier. D-dimer, Ferritin, CRP and Procalcitonin exhibited variable alterations (P<0.05 to P<0.001), more markedly in critically ill patients than in the mild and severe. Biochemical analytes and enzymatic parameters showed elevated levels (P<0.05 to P<0.01) mostly in critically ill category of patients when compared with mild or severe, except total protein and albumin, which remained non-significant. It is concluded that cytokine, chemokines and pro-inflammatory markers, which released in abnormally high concentrations in Covid-19 patients of variable syndrome intensity, are significant indicators of disease severity, progression and success of treatments. As the pharmacological options may vary with the different stages of the disease therefore identifying the correct stage of the disease may be very useful in selecting the best option.

REPORT- Varying pattern of blood gases, ferritin, procalcitonin, C-Reactive protein, interleukin 6 and lactate dehydrogenase in Covid-19 patients suffering from diverse Co-Morbid, suspected cytokine storm and periodic effects of antiviral and steroidal treatments.

This study depicted varying pattern of inflammatory markers and blood gases of selected SARS Covid-19 patients with triggered cytokine storm, during their stay in ICUs, HDUs, on ventilators for 21 days. All were treated with Antiviral (remdesivir), steroid (dexamethason) and antipyretic (paracetamol) medications. Procalcitonin, PCT, C-reactive protein CRP, Interleukin 6 (IL6) and Lactate dehydrogenase (LDH) blood gases pressure (pO2, pCO2), coagulation (D-Dimer DD) and Iron storage proteins (Ferritin Ft) were analyzed by fully automated analyzers. All biomarkers of each patient category was statistically compared with days 1st, 4th, 7th versus 10th, 14th and 17th days and reported as significant where p<0.05, to assess progression, worsening or recovery status. IL6 (P<0.0224, P< 0.0228) and CRP (P<0.0277) exhibited none or mild statistical significance difference, with the exception of Ferritin (P<0.0185; P<0.0088) and D Dimer (P<0.0086), demonstrating slow recovery, revealing stronger cytokine storming assault. LDH, pCO2 and pO2 exhibited variable significance difference when data of earlier days were compared with recovery phase, thus advocating blended treatment or progressing of disease. Analysis confirms overwhelming pathogenesis of SARS Covid-19 distinctive cytokine storm, which needed to be cautiously monitored as infection progressed using pro-inflammatory biomarkers as indicators of recovery or worsening of the disease.

Bi-allelic Loss-of-Function Variants in DNMBP Cause Infantile Cataracts.

Infantile and childhood-onset cataracts form a heterogeneous group of disorders; among the many genetic causes, numerous pathogenic variants in additional genes associated with autosomal-recessive infantile cataracts remain to be discovered. We identified three consanguineous families affected by bilateral infantile cataracts. Using exome sequencing, we found homozygous loss-of-function variants in DNMBP: nonsense variant c.811C>T (p.Arg271∗) in large family F385 (nine affected individuals; LOD score = 5.18 at θ = 0), frameshift deletion c.2947_2948del (p.Asp983∗) in family F372 (two affected individuals), and frameshift variant c.2852_2855del (p.Thr951Metfs∗41) in family F3 (one affected individual). The phenotypes of all affected individuals include infantile-onset cataracts. RNAi-mediated knockdown of the Drosophila ortholog still life (sif), enriched in lens-secreting cells, affects the development of these cells as well as the localization of E-cadherin, alters the distribution of septate junctions in adjacent cone cells, and leads to a ∼50% reduction in electroretinography amplitudes in young flies. DNMBP regulates the shape of tight junctions, which correspond to the septate junctions in invertebrates, as well as the assembly pattern of E-cadherin in human epithelial cells. E-cadherin has an important role in lens vesicle separation and lens epithelial cell survival in humans. We therefore conclude that DNMBP loss-of-function variants cause infantile-onset cataracts in humans.

Role of ALAD and VDR genotypes on the association of low blood lead level with serum uric acid and blood pressure in automobile paint workers of Karachi, Pakistan.

Lead is an environmental pollutant having nephrotoxic effects even at low level. Its continuous exposure is associated with increased serum uric acid level that resulting in renal insufficiency. This research was conducted to see the effects of delta-aminolevulinic acid dehydratase (ALAD) and vitamin D receptor (VDR) genotypes on biochemical parameters and blood pressure (BP) of automobile workers having low blood lead level (BLL) with continuous lead exposure. Automobile paints workers with ALAD 1-2 genotype showed the positive association of BLL with diastolic BP (p<0.05) whereas, a genotypic combination of ALAD 1-2/VDR BB showed the negative association of serum uric acid with BLL (p<0.05). Similarly negative effects of VDR BB genotype (p<0.01) and ALAD 1-2 genotype (p<0.05) were observed in the association of serum uric acid with BLL at the mean age ≥30 years. This suggests that automobile paint workers having ALAD 1-2 genotypes are at the risk of increased diastolic BP. The research also foretells that combination of ALAD 1-2/VDR BB may play a significant role against lead induced nephrotoxicity at low BLL with continuous lead exposure.

Horizontal gene transfer of stress resistance genes through plasmid transport.

The horizontal gene transfer of plasmid-determined stress tolerance was achieved under lab conditions. Bacterial isolates, Enterobacter cloacae (DGE50) and Escherichia coli (DGE57) were used throughout the study. Samples were collected from contaminated marine water and soil to isolate bacterial strains having tolerance against heavy metals and antimicrobial agents. We have demonstrated plasmid transfer, from Amp(+)Cu(+)Zn(-) strain (DGE50) to Amp(-)Cu(-)Zn(+) strain (DGE57), producing Amp(+)Cu(+)Zn(+) transconjugants (DGE(TC50→57)) and Amp(+)Cu(-)Zn(+) transformants (DGE(TF50→57)). DGE57 did not carry any plasmid, therefore, it can be speculated that zinc tolerance gene in DGE57 is located on chromosome. DGE50 was found to carry three plasmids, out of which two were transferred through conjugation into DGE57, and only one was transferred through transformation. Plasmid transferred through transformation was one out of the two transferred through conjugation. Through the results of transformation it was revealed that the genes of copper and ampicillin tolerance in DGE50 were located on separate plasmids, since only ampicillin tolerance genes were transferred through transformation as a result of one plasmid transfer. By showing transfer of plasmids under lab conditions and monitoring retention of respective phenotype via conjugation and transformation, it is very well demonstrated how multiple stress tolerant strains are generated in nature.

Aberrant splicing of an alternative exon in the Drosophila troponin-T gene affects flight muscle development.

During myofibrillogenesis, many muscle structural proteins assemble to form the highly ordered contractile sarcomere. Mutations in these proteins can lead to dysfunctional muscle and various myopathies. We have analyzed the Drosophila melanogaster troponin T (TnT) up1 mutant that specifically affects the indirect flight muscles (IFM) to explore troponin function during myofibrillogenesis. The up1 muscles lack normal sarcomeres and contain "zebra bodies," a phenotypic feature of human nemaline myopathies. We show that the up(1) mutation causes defective splicing of a newly identified alternative TnT exon (10a) that encodes part of the TnT C terminus. This exon is used to generate a TnT isoform specific to the IFM and jump muscles, which during IFM development replaces the exon 10b isoform. Functional differences between the 10a and 10b TnT isoforms may be due to different potential phosphorylation sites, none of which correspond to known phosphorylation sites in human cardiac TnT. The absence of TnT mRNA in up1 IFM reduces mRNA levels of an IFM-specific troponin I (TnI) isoform, but not actin, tropomyosin, or troponin C, suggesting a mechanism controlling expression of TnT and TnI genes may exist that must be examined in the context of human myopathies caused by mutations of these thin filament proteins.

Loop 1 of transducer region in mammalian class I myosin, Myo1b, modulates actin affinity, ATPase activity, and nucleotide access.

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.

Troponin I is required for myofibrillogenesis and sarcomere formation in Drosophila flight muscle.

Myofibrillar proteins assemble to form the highly ordered repetitive contractile structural unit known as a sarcomere. Studies of myogenesis in vertebrate cell culture and embryonic developmental systems have identified some of the processes involved during sarcomere formation. However, isoform changes during vertebrate muscle development and a lack of mutants have made it difficult to determine how these proteins assemble to form sarcomeres. The indirect flight muscles (IFMs) of Drosophila provide a unique genetic system with which to study myofibrillogenesis in vivo. We show in this paper that neither sarcomeric myosin nor actin are required for myoblast fusion or the subsequent morphogenesis of muscle fibres, i.e. fibre morphogenesis does not depend on myofibrillogenesis. However, fibre formation and myofibrillogenesis are very sensitive to the interactions between the sarcomeric proteins. A troponin I (TnI) mutation, hdp(3), leads to an absence of TnI in the IFMs and tergal depressor of trochanter (TDT) muscles due to a transcript-splicing defect. Sarcomeres do not form and the muscles degenerate. TnI is part of the thin filament troponin complex which regulates muscle contraction. The effects of the hdp(3) mutation are probably caused by unregulated acto-myosin interactions between the thin and thick filaments as they assemble. We have tested this proposal by using a transgenic myosin construct to remove the force-producing myosin heads. The defects in sarcomeric organisation and fibre degeneration in hdp(3) IFMs are suppressed, although not completely, indicating the need for inhibition of muscle contraction during muscle development. We show that mRNA and translated protein products of all the major thin filament proteins are reduced in hdp(3) muscles and discuss how this and previous studies of thin filament protein mutants indicate a common co-ordinated control mechanism that may be the primary cause of the muscle defects.