Pro Gene Research Articles

Genomic Sequencing and Analysis of Enzootic Nasal Tumor Virus Type 2 Provides Evidence for Recombination within the Prevalent Chinese Strains.

In recent years, the clinical cases of ENTV-2 infection have increased and become prevalent in several provinces of China. In this study, we reported the occurrence of ENTV-2 in one goat farm in Chongqing, southwest China. The complete genome of an emerged ENTV-2 isolate (designated as CQ2) was sequenced with 7468 bp in length. Phylogenetic analysis revealed that ENTV-2 consisted of two main lineages. Lineage 1 was composed of Chinese strains and could be subdivided into five sublineages. CQ2 and the other six recent isolates from China were clustered in sublineage 1.5; however, CQ2 was significantly different from the other six isolates. Furthermore, recombination analysis suggested that CQ2 might be a recombinant variant derived from sublineage 1.5 and sublineage 1.2 strains, with the recombination region in areas of pro and pol genes. In conclusion, we sequenced and analyzed the complete genome of a potential ENTV-2 recombinant, which may contribute to our understanding of the genetic variation and evolution of ENTV-2 in China.

Abstract PO4-24-01: Comprehensive Molecular and Immunological Characterization of Invasive Ductal Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease and characterized by poor outcomes with a lack of targeted therapies. A comprehensive analysis of the molecular and immune landscape of invasive ductal TNBC can help improve our understanding of TNBC biology and identify novel targets and/or pathways for better management of this disease. Here, we characterized the molecular and immune signature of invasive ductal (ID) TNBC. Methods: 13,036 BC samples (ID TNBC, n=392; ID non-TNBC, n=927) were analyzed by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). A total of 10 pro-apoptotic (BAX, BAK1, BID, BAD, BIK, BCL2L11, BMF, HRK, PMAIP1, BBC3) and 6 anti-apoptotic (BCL2, BCL2L1, BCL2L2, MCL1, BCL2A1, BCL2L10) BCL2 family genes were analyzed. Immune cell fractions were calculated by deconvolution of WTS: Quantiseq. Real world overall survival (OS) and treatment-associated survival was extracted from insurance claims and calculated from tissue collection or treatment start to last contact using Kaplan-Meier estimates. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q < 0.05). Results: ID TNBC had a higher frequency of TP53, RB1, NF1, PIK3R1, NOTCH1, CREBBP, BRCA1, FANCI, and HRAS mutations (Table 1), NOTCH2 (4.3% vs 0.47%), EGFR (3.4% vs 0%), NFIB (4% vs 0.6%), MYB (3.5% vs 0.5%), CCNE1 (4.4% vs 1.1%) and AKT2 (2.4% vs 0.3%) copy number alterations, and NOTCH2 (1.6% VS 0.2%) fusion (all p < 0.05) compared to invasive ductal non-TNBC. Analysis of inferred immune cell infiltrates showed that ID TNBC had increased infiltration of Tregs (2% vs 1.7%), DC (3.2% vs 2.4%), CD8 T cells (0.5% vs 0.1%), and M1 macrophages (M) (3.4% vs 2.9%), but decreased infiltration of B cells (4.5% vs 6%) and M2M (2.9% vs 4.5%) (all p < 0.05). ID TNBC had increased T cell inflamed score (23 vs -9, p < 0.05), IFNy score (-0.24 vs -0.34, p < 0.05), MHC class I genes (HLA-A, HLA-B, TAP1, TAP2, FC: 1.1-1.5, all p < 0.05), immune checkpoint genes (CD274, PDCD1, CTLA4, PD-L2, FOXP3, IDO, FC: 1.1-2, all p < 0.05) and PD-L1 protein expression (SP142: 49.8% vs. 28.4%; 22c3: 41.7% vs. 16.8%, all p < 0.05). ID TNBC had differential expression of BCL2 family genes (upregulation: BAK1, BID, MCL1, BCL2A1, BCL2L10, FC: 1.1-2.0; downregulation: BAD, BIK, BBC3, BCL2, BCL2L1, BCL2L2, FC: 1.2-2.8, all p < 0.05) compared to invasive ductal non-TNBC. ID TNBC was associated with worse OS compared to ID non-TNBC (mOS: 24.5 vs 54.6 months; HR: 0.5, 95% CI 0.47-0.57; p < 0.00001) but trend towards better survival with pembrolizumab (mOS: 29.4 vs 8.8 month; HR: 0.67, 95% CI 0.28-1.5; p=0.3) treatment. No significant OS difference was noted for by high vs. low pro and anti-apoptotic BCL2 genes except for anti-apoptotic BCL2A1 (higher expression associated with better OS (mOS: 5.7 months, HR: 0.78, 95% CI 0.6-0.99; p=0.047). Conclusion: These data indicate that ID TNBC is associated with distinct mutational profile compared to non-TNBC, has increased T cell inflamed score, IFNy score, MHC class I and immune checkpoint gene expression, and differential immune cell infiltration. Interestingly, TNBC was associated with increased M1 and decreased M2M. There is evidence to suggest that transcriptomically defined M1 high tumors are clinically aggressive and have worse OS in TNBC. BCL2 family genes are not prognostic for TNBC outcomes except BCL2A1, which needs further prospective validation. Table 1: Mutation frequency in invasive ductal TNBC and invasive ductal non-TNBC Citation Format: Pooja Advani, Sachin Deshmukh, Sharon Wu, Jacob Andring, Joanne Xiu, Alex Farrell, Jose Leone, Priya Jayachandran, Stephanie Graff, Matthew Oberley, George Sledge Jr, Asher Chanan-Khan. Comprehensive Molecular and Immunological Characterization of Invasive Ductal Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-01.

Leishmanicidal and immunomodulatory activity of Terminalia catappa in Leishmania amazonensisin vitro infection

Leishmaniases are infectious-parasitic diseases that impact public health around the world. Antileishmanial drugs presented toxicity and increase in parasitic resistance. Studies with natural products show an alternative to this effect, and several metabolites have demonstrated potential in the treatment of various diseases. Terminalia catappa is a plant species with promising pharmaceutical properties. The objective of this work was to evaluate the therapeutic potential of extracts and fractions of T. catappa on Leishmania amazonensis and investigate the immunomodulatory mechanisms associated with its action. In anti-Leishmania assays, the ethyl acetate fraction exhibited activity against promastigotes (IC50 86.07 ± 1.09 μg/mL) and low cytotoxicity (CC50 517.70 ± 1.68 μg/mL). The ethyl acetate fraction also inhibited the intracellular parasite (IC50 25.74 ± 1.08 μg/mL) with a selectivity index of 20.11. Treatment with T. catappa ethyl acetate fraction did not alter nitrite production by peritoneal macrophages stimulated with L. amazonensis, although there was a decrease in unstimulated macrophages treated at 50 μg/mL (p = 0.0048). The T. catappa ethyl acetate fraction at 100 μg/mL increased TNF-α levels (p = 0.0238) and downregulated HO-1 (p = 0.0030) and ferritin (p = 0.0002) gene expression in L. amazonensis-stimulated macrophages. Additionally, the total flavonoid and ellagic acid content for ethyl acetate fraction was 13.41 ± 1.86 mg QE/g and 79.25 mg/g, respectively. In conclusion, the T. catappa ethyl acetate fraction showed leishmanicidal activity against different forms of L. amazonensis and displayed immunomodulatory mechanisms, including TNF-α production and expression of pro and antioxidant genes.

Envelope Recombination: A Major Driver in Shaping Retroviral Diversification and Evolution within the Host Genome.

Endogenous retroviruses (ERVs) are integrated into host DNA as the result of ancient germ line infections, primarily by extinct exogenous retroviruses. Thus, vertebrates' genomes contain thousands of ERV copies, providing a "fossil" record for ancestral retroviral diversity and its evolution within the host genome. Like other retroviruses, the ERV proviral sequence consists of gag, pro, pol, and env genes flanked by long terminal repeats (LTRs). Particularly, the env gene encodes for the envelope proteins that initiate the infection process by binding to the host cellular receptor(s), causing membrane fusion. For this reason, a major element in understanding ERVs' evolutionary trajectory is the characterization of env changes over time. Most of the studies dedicated to ERVs' env have been aimed at finding an "actual" physiological or pathological function, while few of them have focused on how these genes were once acquired and modified within the host. Once acquired into the organism, genome ERVs undergo common cellular events, including recombination. Indeed, genome recombination plays a role in ERV evolutionary dynamics. Retroviral recombination events that might have been involved in env divergence include the acquisition of env genes from distantly related retroviruses, env swapping facilitating multiple cross-species transmission over millions of years, ectopic recombination between the homologous sequences present in different positions in the chromosomes, and template switching during transcriptional events. The occurrence of these recombinational events might have aided in shaping retroviral diversification and evolution until the present day. Hence, this review describes and discusses in detail the reported recombination events involving ERV env to provide the basis for further studies in the field.

Protective role of hispolon and its derivatives against apoptosis in cortical neurons induced by electromagnetic radiation from 4G mobile phone.

Electromagnetic radiation (EMR) from wireless devices, particularly mobile phones, is a potentially growing public health concern. In this study, the neuronal effects of EMR on primary cortical neurons (PCNs) from neonatal rat cerebral cortex and the protective role of hispolon (HIS) and its derivatives were investigated as a measure of cranial exposure during mobile phone use.PCNs were isolated and cultured from day-old neonatal rats, then exposed for 2 h to EMR emitted by a mobile phone operating at a frequency of 2100 MHz with 1.6 W/Kg specific absorption rate (SAR) in call-answered mode treated with HIS and its derivatives. The induction of apoptosis through modulation of pro and anti-apoptotic genes via mitochondrial pathway and the protection by the test compounds was assessed. Pyrazole derivatives decreased apoptosis by modulating the levels of pro and anti-apoptotic genes by reducing the levels of reactive oxygen species (ROS) via mitochondrial damage, which was observed in the EMR exposed PCNs. The pyrazole compounds were found to have antioxidative and anti-apoptotic properties. Thus, the neuroprotective mechanisms of the pyrazole derivatives can be investigated further, which may make them appropriate as lead compounds in developing neuroprotective formulations.

Molecular detection of mixed infection with peste des petits ruminants and retroviruses in Egyptian sheep and goats.

Peste des petits ruminants (PPR) is a contagious viral disease causing massive economic loss to animal industries in endemic countries including Egypt. Although a vaccine is available, coinfections can overwhelm the animal immune system and interfere with vaccine protection. Small ruminant retrovirus (SRR), including enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV), is responsible for coinfections with PPR. Investigation of clinical cases in this study confirmed the presence of PPR virus by RT-PCR among four flocks. Sequence of five PPR amplicons revealed that all strains had 100% aa similarity and belonged to lineage IV. In addition, these strains had 98-99% nt similarity with all previous Egyptian and African strains from Sudan (MK371449) and Ethiopia (MK371449). Illumina sequencing of a representative sample showed a genome of 5753 nt compatible with ENT-2 virus with 98.42% similarity with the Chinese strain (MN564750.1). Four ORFs representing gag, pro, pol, and env genes were identified and annotated. Pro gene was highly stable while gag, pol, and env showed eight, two, and three aa differences with the reference strains. Sanger sequencing revealed that two amplicons were ENT-2 virus, and one was JSRV. ENT-2 sequences had 100% similarity with KU258870 and KU258871 reference strains while JSRV was 100% similar to the EF68031 reference strain. The phylogenetic tree showed a close relationship between the ENT of goats and the JSRV of sheep. This study highlights the complexity of PPR molecular epidemiology, with SRR that was not molecularly characterized previously in Egypt.

Identification and molecular characterization of simian endogenous retrovirus in Macaca fascicularis and Macaca nemestrina from captive breeding facilities in Bogor, Indonesia.

Background and Aim:Endogenous retroviruses (ERVs) found in all vertebrates, including non-human primates (NHPs), are known to be genetically inherited. Thus, recent studies have explored ERVs for human immunodeficiency virus vaccine development using human ERV (HERV) due to the hypervariability of exogenous retroviruses which cause conventional vaccines to be ineffective. HERV was also found to be able to induce an immune response in cancer patients. This study aimed to identify and molecularly characterize ERVs from Indonesian NHPs: Macaca fascicularis and Macaca nemestrina. Then, we described the phylogenetic relationship of these isolates with those of the simian ERVs (SERVs) characterized in other species and countries.Materials and Methods:First, 5 mL of whole blood samples was taken from 131 long-tailed macaques and 58 pig-tailed macaques in captive breeding facilities at Bogor, Indonesia, for DNA extraction. Next, the DNA samples were screened using the SYBR Green real-time polymerase chain reaction (PCR) technique with specific primers for env (simian retroviruses [SRV]1-5 7585U19 and SRV1-5 7695L21). Positive SERV results were those with cycle threshold (CT) values < 24 (CT < 24) and melting temperature (TM) ranges of 80°C–82°C. Then, whole-genome nucleotide sequences from two pig-tailed macaques samples detected as positive SERV were generated using a nucleic acid sequencing technique which utilized the walking primer method. Subsequently, the sequences were analyzed using bioinformatics programs, such as 4Peaks, Clustal Omega, and BLAST (NCBI). Subsequently, a phylogenetic tree was constructed using the neighbor-joining method in MEGA X.Results:SYBR Green real-time PCR amplification results indicated that SERV (Mn B1 and Mn B140910)-positive samples had CT values of 22.37–22.54 and TM of 82°C. Moreover, whole-genome sequences resulted in 7991 nucleotide sequences, comprising long terminal repeat, gag, pro, pol, and env genes identical between the sequenced samples. Furthermore, the phylogenetic tree results indicated that both samples from M. nemestrina had 99%–100% nucleotide identities to the Mn 92227 sample identified at the National Primate Center University of Washington (NaPRC UW) which was imported from Indonesia in 1998, confirmed as a novel SERV strain. The phylogenetic tree results also indicated that although SERV whole-genome nucleotide and env amino acid sequences were clustered with SRV-2 (identity values of 82% and 79%, respectively), they had a 99%–100% nucleotide identity to Mn 92227. Meanwhile, the gag, pro, and pol amino acids were clustered with SRV-1, SRV-3, SRV-4, SRV-5, SRV-8, and SERV/1997, with 82% and 88% identity values.Conclusion:Based on the SYBR Green real-time PCR profiles generated, similarities with Mn 92227 were observed. Subsequent phylogenetic analysis confirmed that both samples (Mn B1 and Mn B140919) from pig-tailed macaques in the country of origin were novel SERV strains at NaPRC UW. Therefore, it could be used in biomedical research on ERVs.

Role of Proximal Tubule DPP4 in Inflammation and Kidney Disease Progression

Obesity and overweight leads to an entity called obesity related kidney disease (ORKD), which is a major risk factor for chronic kidney disease (CKD) progression to End Stage Renal Disease and/or dialysis. ORKD is characterized by proximal tubule brush border vacuolization and injury, basement membrane thickening, glomerular accumulation of fat globules and glomerulomegaly. We have developed a mouse model of Western diet (high fat and high sucrose)‐induced obesity that manifests all the features of ORKD. In addition, our lab observed that obese mice fed a Western diet, manifest early hyperfiltration (3 months into feeding). It is later followed by reduction in glomerular filtration rate (GFR, beginning at 18 months of age), and this pattern mimics human diabetic kidney disease progression. Our lab also observed that in ORKD, there is increased Dipeptidyl Peptidase 4 (DPP4) activity in the kidney, especially the proximal tubules and proximal tubule specific deletion of DPP4 improves the GFR. Lastly, our lab demonstrated that there is a major role for inflammation in the obese kidney and DPP4 inhibitors mitigate the inflammation. However, it is not known if and how proximal tubule DPP4 contributes to inflammation and pathophysiological changes in ORKD. Therefore, we hypothesized that proximal tubule DPP4 mediates inflammation and contributes to the pathophysiological changes in ORKD, thereby identifying DPP4 as a target to prevent CKD progression.MethodsProximal tubule specific DPP4fl/fl;SGLT2Cre+ (PT DPP4KO) and DPP4fl/fl;SGLT2Cre‐ (PT DPP4WT) mice were fed a WD and control chow (CD) for 6 mths, 15 mths and till sacrifice (18 to 27 mths). Inflammation was assessed both by flow cytometry assessment of kidney immune cells and expression of pro and anti‐inflammatory genes and proteins).ResultsThere were more pro‐inflammatory as well as DPP4 expressing macrophages in the WD‐fed WT group compared to the KO group (p&lt;0.05). There was also activation of the T‐cells in particular the CD8s which was suppressed in the KO group (p&lt;0.05). Deep sequencing of kidney mRNA revealed activation of pro‐inflammatory cytokines including Th1, Th17, M1 macrophage‐based cytokines and TLR4 and ICAM‐1. The KO group showed improvement in both TLR4 and ICAM‐1 (p&lt;0.05). At the protein level, there was an increase in pro‐fibrotic (Desmin) and pro‐inflammatory (ERK) proteins in the WD‐fed WT groups and phosphorylation of ERK was suppressed in the KO group (p&lt;0.05). When inflammation was assessed across the months it seemed that the earliest changes were in the growth pathways and beta‐oxidation of lipids, followed by the more inflammatory genes later in the course of the disease.ConclusionWD feeding led to hyperfiltration followed by a decline in GFR that was partly mitigated by PT deletion of DPP4 and this was associated with improved kidney histology and less fibrosis. WD feeding in WT mice was associated with increase in expression of several pro inflammatory genes and TLR4 and ICAM 1 were normalized in KO mice. DPP4 protein expression in kidney was associated with increased phosphorylation of ERK and this was suppressed in KO mice. Overall, these data suggest that suppression of early activation of DPP4 in ORKD may slow down the progression of CKD.

Fabrication and assessment of folic acid conjugated-chitosan modified PLGA nanoparticle for delivery of alpha terpineol in colon cancer

The objective of this study was to fabrication of α-terpineol-PLGA nanoparticles coated with folic acid-chitosan (αT-PCF-NPs) as well as evaluates their anticancer effects. αT-PCF-NPs were synthesized using the nanoprecipitation method and characterized by Dynamic light scattering (DLS), zeta potential (ZP), scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) analysis. Folic acid (FA) binding rate and entrapment efficiency of α-T were assessed by HPLC method. MTT assay was performed for cytotoxicity assessment. Quantitative polymerase chain reaction (qPCR) analysis, acridine orange and propodium iodide (AO/PI) staining and cell cycle analysis were done to assess the pro-apoptotic properties of αT-PCF-NPs. Molecular analysis for angiogenesis and antioxidant properties and murine colon cancer model for antitumor effects of αT-PCF-NPs were used. The % FA-binding and encapsulation efficiency of α-T in αT-PCF-NPs (particle size of 263.95 nm, polydispersity index (PDI) of 0.25, and surface charge of +38.20 mV) was reported to be 67% and 88.1% respectively. The higher inhibitory effect of αT-PCF-NPs on cancer cells compared to HFF cells was confirmed. The pro-apoptotic effect of αT-PCF-NPs was showed by increased SubG1 phase cells, AO/PI staining results and up and down regulation Bax and Bcl-2 as pro and anti-apoptotic genes in HT-29 cells. Antioxidant (SOD) and angiogenesis genes (VEGF and VEGF-R) were inhibited by αT-PCF-NPs exposure in HT-29 cells and also decreased the size of murine tumors was confirmed in exposure of αT-PCF-NPs. αT-PCF-NPs can be considered as a promising anticancer drug for colon cancer.

Production of recombinant SARS-CoV-2 3CL-protease: The key for the development of protease inhibitors screening kit in search of potential herb cure for COVID-19

Coronavirus disease-19 (COVID-19) pandemic caused millions of deaths and socio-economics damage worldwide. Corresponding to this, many studies on antiviral drugs exploration are rising to investigate the potential of drug compounds that can repress the replication of the SARS-CoV-2 virus by targeting its main protease named 3-chymotrypsin like protease (3CLpro). Without 3CLpro splicing, polyproteins of SARS-CoV-2 will not function to form new virions. We conducted an in silico thermodynamic study, and found several already known compounds from natural sources such as lovastatin, quinidine, and quinine were potential in inhibiting 3CLpro. However, most of the findings still need further wet-lab experiments to confirm their activity against it. To facilitate rapid screening of protease inhibitors, we aim to develop a screening kit for researchers with focus studies on herbal sources for COVID-19 treatments. Hence, the development of 3CLpro as the target of inhibition screening is essential. Our current work focused on the expression of the SARS-CoV-2 3CL Pro gene harbored by pET-32b(+) in E. coli BL21(DE3) and its purification. The recombinant E.coli were cultivated in LB media at 37 °C with 5 hours IPTG-induced phase followed by crude protein lysate extraction by sonication, and then Ni-NTA purification. A band of recombinant 3CLpro protein (33.8 kDa) detected by SDS PAGE and Western blotting after Ni-NTA purification was specific and clear, showing the right size of our protein interest. A final concentrated protein mixture obtained by 10-kDa membrane filtration displayed >90% purity with a total protein of 2.497 mg mL−1. Further functional assays and inhibitory tests with several natural compounds showed that 3CLpro functioned well as a drug target.

Gestational diabetes is associated with changes in inflammatory gene expression related to human placental macrophages

Gestational diabetes (GDM) is associated with adverse pregnancy outcomes. Metabolic dysfunction with diabetes can alter immune responses. With GDM, changes in the placental immune system are thought to drive pregnancy complications. The objective of this study as to evaluate placental pro and anti-inflammatory gene expression in pregnancies complicated by GDM.

ARMC5 modifies cell redox state to regulate steroidogenesis and lipid metabolism in the adrenal cortex

ARMC5 is a putative tumor suppressor gene that is frequently mutated in primary bilateral macronodular adrenal hyperplasia (PBMAH), a rare cause of Cushing's syndrome. The function of ARMC5 is poorly known, aside the fact that it regulates cell apoptosis and adrenal steroidogenesis in by mechanisms still unknown. Tumor suppressor genes play an important role in oxidative stress. In this study we used as model the adrenocortical carcinoma cell line H295R. In order to investigate ARMC5 response to stress, cells were treated with Menadione and reactive oxygen species (ROS) production was measured by Cellrox assays with flow cytometry. Oxidative response genes and steroidogenic enzymes expression were investigated by qPCR, whereas p38 phosphorylation was investigated by western blotting. ARMC5 protein is differentially regulated in response to menadione-induced stress in H295R adrenocortical cells. Moreover, ARMC5 depletion increases total intracellular ROS production (P < 0.05) and causes an imbalanced transcription of pro and anti-oxidant genes leading to increased phosphorylation of p38 (P < 0.05). ROS production and p38 pathway activation alter steroidogenesis. These effects are partially reversed by the anti-oxidant compound N-acetylcysteine (NAC) or the p38 inhibitor (SB203580). Altogether, this study describes a new function of ARMC5 as regulator of the redox homeostasis in adrenocortical cells.

HIV-1 subtypes and profiles of resistance to protease inhibitors in the Marajo Archipelago (Brazilian Amazon region)

The Marajo Archipelago, located in the rural area of ​​the state of Para (Amazon region of Brazil), is the region with the lowest index of human development in the country, presenting poor health services, which makes adequate epidemiological surveillance of HIV-1 infection difficult. After a serological screening enrolling 1,877 samples, three samples were diagnosed with HIV-1 and the pro gene was sequenced to evaluate the presence of resistance mutations to protease inhibitors. Molecular analyses revealed, for the first time in the region of Marajo Island, the occurrence of HIV-1 subtypes B and D, as well as the presence of transmitted high and intermediate protease inhibitors resistance mutations. The results emphasize the importance of ongoing molecular epidemiological surveillance studies in the Brazilian Amazon region because antiretroviral (ARV) resistance mutations may limit treatment options, and the presence of certain subtypes seems to influence the progression to AIDS, particularly in areas where the entry and spread of the virus can be facilitated by socio-demographic problems that expose the population to sexually transmitted infections.

Single Nucleotide Polymorphisms (SNPs), Phylogeny and Spatiotemporal analysis of pro gene in HIV samples from Pakistan

Background: The increase in incidence of HIV infection with limited treatment options has enhanced the morbidity and mortality in South Asian countries. The mutations, geographical and genetic diversity in Pro gene in HIV positive patients from other South Asian countries can be helpful for treatment options. Aim: To identify mutations and genetic diversity in Pro gene in HIV positive patients in Pakistan and compare the sequences to neighbouring countries China and India. Methods: The HIV protease gene or pro-gene sequence were retrieved from NCBI. The sequence were studied from Pakistan, China and India were analysed for their linkage and genetic mutations through bioinformatics tools, MEGAX and CLUSTALW Results: The phylogenetic analysis of samples for maximum likelihood and mutation from South Asian countries Pakistan, China and India individually displayed variation in sequences of HIV pro gene in all these isolates. Whereas Pakistani isolates have more genetic similarities with the isolates from China than India. Conclusion: The phylogeny analysis depicts there is gradual evolution in viral types and possible entry is through the neighbouring transmission might have been through social connections. The mutations studies provides bases for the novel targets for the drug design and development against HIV. Keywords: HIV, Protease gene, MEGA, Phylogenetic analysis, Pakistan

Polymorphism of Pro and Anti-Inflammatory Cytokine Genes in Children with Legg-Calve-Perthes Disease

Polymorphism of Pro and Anti-Inflammatory Cytokine Genes in Children with Legg-Calve-Perthes Disease

P-PN009. Photobiomodulation promoting recovery from traumatic nerve injury secondary to supracondylar fracture of humerus: A case report

Introduction. Photobiomodulation using low level laser therapy (LLLT-produced by low to mid- powered coherent lasers ranging from 1 mW to 500 mW) has been researched extensively in animal experiments and is shown to enhance nerve healing; however, few clinical studies report effectiveness of photobiomodulation in humans. Recent understanding suggests that LLRT-induced mitochondrial activity leads to increased ATP production, transcription factor induction, pro and anti-inflammatory gene expression causing cell biostimulation, cytokine modulation, growth factors release and increased tissue oxygenation ameliorating nerve injury. This clinical case report aims to highlight clinical nerve injury recovery after LLLT in a 7-year- old girl following a traumatic Gartland type 3 supracondylar fracture of the right humerus sustained in May 2019. Clincal examination of patient NS suggested right median and ulnar axonometsis evidenced by wasting of intrinsic muscles, thenar and clawing of all digits. After undergoing Closed Manual Reduction and K-wiring there was minimal recovery of motor function below the right wrist. Results. Center based physio and occupational therapy was instituted together with home based therapy - NMES (Neuromuscular electrical stimulation) immediately following injury and surgical management. Typically 86-100% of supracondylar fracture associated nerve injuries resolve spontaneously within 6 months with recovery mean time between 2 and 3 months and in this case, there was some recovery (index finger extension) but stagnated at 5 months. At University Malaya Medical Center (UMMC) she underwent 10 sessions of LLLT at the Rehabilitation Medicine clinic, over a 2 week period resulted in remarkable improvement in motor function especially wrist extension (beyond 90 degrees), extension of 3rd to 5th fingers and 5th finger abduction. Conclusion. Our findings suggest that a structured, intensive course of LLLT has the potential to enhance nerve recovery. Further research is required to determine the duration, intensity and dosage of LLLT to streamline prescription and clinical outcomes.

MDM2 promoter polymorphism (rs2279744) and serum estrogen level are associated with increased risk of epithelial ovarian cancer: A case-control study

Introduction: Epithelial ovarian cancer (EOC) is most common malignancy affecting Indian women. One of the transcriptional targets of p53 is human homolog of Murine Double Minute 2 Oncogene (MDM2) which is a crucial negative regulator of p53. Polymorphism in intronic promoter P2 region of MDM2 at position 309 increase binding affinity of specific protein 1, lead to over expression of MDM2 and deflation of p53 pathway. The objectives of study were to find effect of MDM2 SNP309 T>G polymorphism and serum estrogen level on risk of development of EOC and interaction between MDM2 SNP309 T>G and TP53 72 Arg>Pro polymorphism. Materials and Methods: Eighty EOC cases and eighty age matched healthy controls were included in study. MDM2 SNP309 T>G polymorphism was detected by ASO-PCR technique and serum estrogen levels were detected by chemiluminescence immunoassay. Result: MDM2 SNP309 GG genotype was associated with significantly increased risk (OR: 2.86, 95%, CI: 1.07-7.66; P=0.03) and early age onset (P=0.001) of EOC. Serum Estrogen was significantly (PG and TP53 72Arg>Pro gene polymorphism act additively in developing ovarian cancer. Conclusion: This study provides evidence that MDM2 SNP309 T>G promoter polymorphism in association with the higher serum estrogen levels is associated with genetic susceptibility and early age onset of ovarian cancer. Keywords: Epithelial ovarian cancer, MDM2, TP53, Estrogen.

Maternal high-fat diet induces sex-specific changes to glucocorticoid and inflammatory signaling in response to corticosterone and lipopolysaccharide challenge in adult rat offspring

BackgroundMaternal obesity as a result of high levels of saturated fat (HFD) consumption leads to significant negative health outcomes in both mother and exposed offspring. Offspring exposed to maternal HFD show sex-specific alterations in metabolic, behavioral, and endocrine function, as well as increased levels of basal neuroinflammation that persists into adulthood. There is evidence that psychosocial stress or exogenous administration of corticosterone (CORT) potentiate inflammatory gene expression; however, the response to acute CORT or immune challenge in adult offspring exposed to maternal HFD during perinatal life is unknown. We hypothesize that adult rat offspring exposed to maternal HFD would show enhanced pro-inflammatory gene expression in response to acute administration of CORT and lipopolysaccharide (LPS) compared to control animals, as a result of elevated basal pro-inflammatory gene expression. To test this, we examined the effects of acute CORT and/or LPS exposure on pro and anti-inflammatory neural gene expression in adult offspring (male and female) with perinatal exposure to a HFD or a control house-chow diet (CHD).MethodsRat dams consumed HFD or CHD for four weeks prior to mating, during gestation, and throughout lactation. All male and female offspring were weaned on to CHD. In adulthood, offspring were ‘challenged’ with administration of exogenous CORT and/or LPS, and quantitative PCR was used to measure transcript abundance of glucocorticoid receptors and downstream inflammatory markers in the amygdala, hippocampus, and prefrontal cortex.ResultsIn response to CORT alone, male HFD offspring showed increased levels of anti-inflammatory transcripts, whereas in response to LPS alone, female HFD offspring showed increased levels of pro-inflammatory transcripts. In addition, male HFD offspring showed greater pro-inflammatory gene expression and female HFD offspring exhibited increased anti-inflammatory gene expression in response to simultaneous CORT and LPS administration.ConclusionsThese findings suggest that exposure to maternal HFD leads to sex-specific changes that may alter inflammatory responses in the brain, possibly as an adaptive response to basal neuroinflammation.

MicroRNA binding site polymorphism in inflammatory genes associated with colorectal cancer: literature review and bioinformatics analysis.

Inflammation, among environmental risk factors, is one of the most important contributors to colorectal cancer (CRC) development. In this way, studies revealed that the incidence of CRC in inflammatory bowel disease patients is up to 60% higher than the general population. MicroRNAs (miRNAs), small noncoding RNA molecules, have attracted excessive attention due to their fundamental role in various aspects of cellular biology, such as inflammation by binding to the 3'-untranslated regions (3'-UTR) of pro and anti-inflammatory genes. Based on multiple previous studies, SNPs at 3'-UTR can affect miRNA recognition elements by changing the thermodynamic features and secondary structure. This effect can be categorized, based on the number of changes, into four groups, including break, decrease, create, and enhance. In this paper, we will focus on functional variants in miRNA binding sites in inflammatory genes, which can modulate the risk of CRC by both investigating previous studies, regarding miRSNPs in inflammatory genes associated with CRC and recruiting in silico prediction algorithms to report putative miRSNPs in 176 inflammatory genes. In our analysis, we achieved 110 miRSNPs in 3'-UTR of 67 genes that seem good targets for future researches.

Static Magnetic Field Exposure In Vivo Enhances the Generation of New Doublecortin-expressing Cells in the Sub-ventricular Zone and Neocortex of Adult Rats

Static magnetic field (SMF) is gaining interest as a potential technique for modulating CNS neuronal activity. Previous studies have shown a pro-neurogenic effect of short periods of extremely low frequency pulsatile magnetic fields (PMF) in vivo and pro-survival effect of low intensity SMF in cultured neurons in vitro, but little is known about the in vivo effects of low to moderate intensity SMF on brain functions. We investigated the effect of continuously-applied SMF on subventricular zone (SVZ) neurogenesis and immature doublecortin (DCX)-expressing cells in the neocortex of young adult rats and in primary cultures of cortical neurons in vitro. A small (3 mm diameter) magnetic disc was implanted on the skull of rats at bregma, producing an average field strength of 4.3 mT at SVZ and 12.9 mT at inner neocortex. Levels of proliferation of SVZ stem cells were determined by 5-ethynyl-2′-deoxyuridine (EdU) labelling, and early neuronal phenotype development was determined by expression of doublecortin (DCX). To determine the effect of SMF on neurogenesis in vitro, permanent magnets were placed beneath the culture dishes. We found that low intensity SMF exposure enhances cell proliferation in SVZ and new DCX-expressing cells in neocortical regions of young adult rats. In primary cortical neuronal cultures, SMF exposure increased the expression of newly generated cells co-labelled with EdU and DCX or the mature neuronal marker NeuN, while activating a set of pro neuronal bHLH genes. SMF exposure has potential for treatment of neurodegenerative disease and conditions such as CNS trauma and affective disorders in which increased neurogenesis is desirable.