Molecular and Genetic Medicine

The chitin-derived biopolymer chitosan has been shown to possess immunostimulatory properties in several systems and has rendered interest as a candidate adjuvant in vaccine formulations. The mechanisms underlying this effect are not completely understood at the cellular and molecular level, but activation of inflammasome and caspase-1 in antigen presenting cells (APC) was recently suggested to be vital for immune activation. In this study, chitosan-based viscoelastic hydrogel particles of two sizes, 10 μm and 200 μm, were evaluated in regard to cellular uptake and activation of APCs. Macrophages derived from the human cell line THP-1 were shown by flow cytometry and confocal laser scanning microscopy to take up FITC-labelled chitosan particles of both sizes via an active process that could be inhibited by cytochalasin D. To investigate if the viscoelastic chitosan particles cause inflammasome activation, NFκB and IL-1β was measured in THP-1 derived macrophages after 24 h incubation with the chitosan particles with or without priming of the cells with LPS. We found that chitosan particles of both sizes stimulated upregulation of NFκB and IL-1β in the absence of LPS. Finally the dependence of this effect on inflammasome-mediated activation of caspase-1 was assessed. Active caspase-1 was not detected in THP-1- derived macrophages stimulated with chitosan-based viscoelastic hydrogel particles, neither alone nor in combination with LPS. In conclusion, we show that viscoelastic chitosan particles in the size range of 10-200 μm, are taken up by human APCs. Moreover, our study suggests that the chitosan particles stimulate NFκB upregulation and IL-1β secretion through inflammasome activation via a caspase-1 independent pathway.

DNA methylation and nucleic acid biosynthesis are two crucial phenomena for normal chromosomes segregation. From our earlier studies, MTHFR 677T individually and in combination with other gene polymorphisms, micronutrient deficiency and hyperhomocysteinemia was shown to be associated with risk in Down syndrome (DS) mothers. Remethylation and nucleic acid biosynthesis pathways are dependent on the activity of Methylenetetrahydrofolate reductase (MTHFR) and Thymidylate synthase (TYMS) respectively, competing for common substrate molecule 5,10-methelenetetrahydrofolate (5,10-MTHF). Role of MTHFD1 1958 G>A (affecting synthesis of 5,10-MTHF), MTHFR 677 C>T (affecting methylation), TYMS 5'UTR 28 bp repeat polymorphism and TYMS 3'UTR 6bp deletion polymorphism (affecting nucleic acid biosynthesis) in a cohort of 200 case mothers and 187 control mothers (also 146 case triads: mother, father, and child) were studied. We observed a significant association of MTHFR 677 C>T in a co-dominant model (p=0.0428) and dominant model (0.0194) as well as TYMS 5'UTR 28 bp repeat polymorphism in a recessive model (p=0.0005) and dominant model (0.0161). Genetic combination analysis revealed a significant additive effect of certain genotypic combinations (especially combination of MTHFR 677T and TYMS 2R alleles with other alleles or genotypes) in increasing risk. Weak linkage disequilibrium (LD) was observed between TYMS 5' and 3' UTR regions polymorphism in LD analysis. Transmission disequilibrium test (TDT) analysis revealed a consistent trend of preferential allele's transmission from parents. We concluded that genetic interaction of remethylation pathway and the nucleic acid metabolic pathway was significantly associated with risk factors for DS childbirth. However, replication studies are required to validate our observation in the population.

Viral genes may act as oncogenes and interact with intracellular proteins, which are related to oncogenesis and tumor growth. Autocrining CXCL12 in growing tumor tissue plays critical roles in modulating cell proliferation and survival through its receptor CXCR4 and CXCR7. Recently, a number of research reports indicated that CXCR7 displays high expression in many kinds of tumor tissues and cell lines and that high -expressed CXCR7 may be involved in cell survival and migration. To date, CXCR7’s biological function and signaling in cancer pathogenesis is unclear. In this study, we first found there are higher expression of CXCR7 in both lymphocyte nodes and EBV positive lymphoma cell line IB4. The results further suggested that high-expressed CXCR7 plays a role in CXCL12 mediated anti-apoptosis and inhibition of CXCR7 through CXCR7 inhibitor CCX771 significantly attenuated the function of CXCL12 on anti-apoptosis. Furthermore, we observed that CXCR7 inhibitor directly reduced protection of endogenous CXCL12 from apoptosis in EBV positive lymphoma cell line. Finally, the results revealed that CXCL12 activated CXCR7 trafficking to membrane and interaction of CXCR7 and β-arrestin-2 during anti-apoptosis process. The study suggested that the high expressed CXCR7 may have effect on anti-apoptosis and inhibitor of CXCR7 may become potential target for cancer therapy.

Cranberry products have long been used to treat urinary tract infections. It is believed that the A-type proanthocyanidins in cranberries contribute to this function. Peanut is one of the other, few food sources that primarily contain A-type proanthocyanidins. The skin on the outside of the peanut kernels (testa), which is treated as an agriculture waste product, contains high levels of A-type proanthocyanidins. In this study, an HPLC diol column separation method and MALDI-TOF MS were used to characterize and compare the proanthocyanidin compositions of peanut skins and cranberries. MALDI-TOF MS in linear mode was able to detect a group of proanthocyanidins with DP (degree of polymerization) 10 in peanut skin extract, but was only able to detect DP 8 in cranberry extract. The reflectron mode showed clusters of clear narrow peaks at DP 7 in peanut skin extract, while the highest DP resolved for cranberry extract was only 3 in reflectron mode. This might be due to the low response intensity of the cranberry samples with the current cleanup method and the matrix. Based on the resolved peaks in reflectron mode, pPeanut skins and cranberries have similar proanthocyanidins composition; they contain both A-type and B-type proanthocyanidins, with the A-type being predominant. This result may inspire future studies on the comparison of biological functions between peanut skins and cranberries and further comparison of their polymeric proanthocyanidin composition.

Background: Pharmacogenetics is the study of genetic influence on pharmacological response. Pharmacogenetic testing serves to identify the presence of genetic variants which may affect pharmacological outcomes, and allows for the selection of pharmacological therapy based on a patient's specific genetic make-up. Therefore, it has the potential to become an invaluable resource in certain fields of medicine to provide patient-tailored pharmacotherapy to patients.
Objective: To determine the clinical relevance of pharmacogenetic testing.
Methods: A systematic review was conducted from September 2013- November 2015 using the EMBASE and EBSCO host databases, identifying English language Cochrane reviews, controlled clinical trials, randomized control trials, meta-analyses and systematic reviews conducted on humans. Search terms pertaining to pharmacogenetic testing in the following medication classes: cardiovascular, oncologic, pain management, antiretroviral, and antidepressant were used. Selected articles were evaluated and assigned ratings based on the level of evidence present. A rating of "A" was assigned for high level of evidence, "B" for moderate level of evidence, and "C" for minimal level of evidence.
Results: The literature search resulted in a total of twenty-one selected articles of interest. Of these articles, seven were identified with an evidence rating of "A" and four articles with an evidence rating of "B".
Conclusion: Pharmacogenetic testing is relevant to clinical practice in certain situations. Its use provides health care providers with additional information which may enable them to treat patients more efficiently by preventing adverse reactions and anticipating therapeutic responses. A lack of prospective randomized control trials, ethical concerns, and a lack of provider knowledge pertaining to pharmacogenetic testing remain as barriers to routine pharmacogenetic testing in clinical practice. Despite these barriers, the future of pharmacogenetic testing is promising and expected to be welcomed by those whom are concerned with providing optimal pharmaceutical care.

Background: Hemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII gene (F8C). Molecular testing for the factor VIII gene is difficult due to its large size. More than 1000 different mutations have been described in factor VIII gene. In this study we have investigated the factor VIII gene mutations in Saudi Arabian population. Methods: For genotyping factor VIII cohorts of 110 samples from Saudi Arabian patients undergoing treatment for hemophilia A were collected. All patients were tested for factor VIII coagulant activity on Behring Coagulation System. Genomic DNA was isolated from blood on MagNapure system. Screening for inv-1 was done by multiplex PCR method, and inv-22 was done by ligation (inverse) PCR method. DNA sequencing was performed by Sanger method for all 26 exons of factor VIII gene. PCR products were sequenced on ABI 3500 Genetic analyzer. For molecular simulations we have used softwares such as CHARMM and GROMACS v4.0.527. In order to predict the possible impact of a variation on the function of factor VIII gene the online tools Polyphen 2, and SIFT were used. Results: Out of 110 cases screened, 2 patients were positive (affected) for inv-1 and 15 patients were positive (12 affected and 3 carriers) for inv-22. Out of 32 cases sequenced for coding exons, 2 novel mutations were found, one novel missense mutation c.355G>C, p. (A119P) in exon 3, and another novel frame shift mutation c.6482delC, p.(P2161Lfs*25) in exon 23. Also known mutations such as, c.409 A>C, p. (T137P) in 2 individual patients in exon 4, another known mutation c.1804C>T, p.(R602*) in 1 patient in exon 12 were found. Genotype-phenotype correlations and computer prediction analysis on these novel mutations and the secondary structure analysis of the factor VIII protein were performed, and compared with the predicted native proteins. Conclusions: These novel mutations in factor VIII gene and molecular dynamic simulation results to appropriately predict the deleterious effects of these mutations are presented in this study. In addition, for the native and mutant proteins models, the amino acid residues and its secondary structures were determined. Our In-silico study suggests that these mutations have significant impact on the structure and function of the factor VIII protein.

The present study was aimed to investigate the role of Wnt/β-catenin pathway in peripheral blood mononuclear cells (PBMCs) derived from patients with Facioscapulohumeral muscular dystrophy (FSHD). Our results showed severe impairment in β-catenin signaling pathway in the PBMCs of FSHD patients. We suppose the aberration of β-catenin expression in the PBMCs of FSHD patients may greatly reflect the severity of neuromuscular degeneration of FSHD pathology per se, which may occurred due to the loss of β-catenin and its prosurvival and regenerative roles against the inflammatory/oxidative/apoptotic cascades of FSHD pathology. These preliminary data from four consanguineous FSHD patients may offer, at least in partial, putative molecular cues underlying the pathophysiology of FSHD.

Objectives: This study examined in vitro responses of fibroblast cellular respiration to lipoic acid, thiamine and carnitine in patients with dihydrolipoyl dehydrogenase (DLD) deficiency. This disorder impairs cellular bioenergetics and these compounds are used to improve clinical manifestations of the disease. The study aimed to utilize mitochondrial O2 consumption as a surrogate biomarker for examining cellular responses to metabolic therapies.

Methods: Cultured fibroblasts from three patients were treated with therapeutic concentrations of the compounds for 24 hours. Cells were then harvested and processed for measuring respiration using phosphorescence oxygen analyzer. Patients 1 and 2 were severely symptomatic infants with homozygous c.1436A>T mutation in the DLD gene. Patient-3 was a mildly symptomatic adolescent with homozygous c.685G>T mutation.

Results: The rate of respiration (mean ± SD, n=6, μM O2 min-1/107 cells) in fibroblasts from a normal infant was 9.3 ± 1.6, in fibroblasts from Patient-1 was 5.1 ± 0.9 (p=0.001), in fibroblasts from Patient-2 was 7.4 ± 1.4 (p=0.051), and in fibroblasts from Patient-3 was 10.3 ± 3.3 (p=0.836). In normal fibroblasts, respiration decreased by the thiamine (p=0.012) and carnitine (p=0.023) treatments. In Patient-1, respiration increased by the lipoic acid (p<0.002), thiamine (p<0.001), and carnitine (p=0.018) treatments; this patient clinically responded to thiamine. In Patient-2, respiration decreased by the thiamine (p=0.026) and carnitine (p=0.008) treatments; this patient did not respond to these drugs. In Patient-3, respiration increased by the carnitine (p=0.012) treatment; the patient clinically responded to carnitine.

Conclusions: The results show cellular respiration is a suitable biomarker for the disease. The significance of using this tool to assess responses to therapies requires further studies.

Carotenoids are valuable pigments of commercial interest for various health benefits. There is rising demand for natural carotenoids from microorganisms, although the majority of industrially produced carotenoids are currently chemically synthesized. Rhodosporidium toruloides is oleaginous red yeast, which can produce large amounts of carotenoids and lipids simultaneously. It is also able to assimilate waste substrates as a carbon source. In this review we propose that Rhodosporidium toruloides's high lipid accumulating ability, is linked to its ability to also produce carotenoids. We provide details of the production and regulation of carotenoids and lipids biosynthesis in this yeast, which appear to be in sync. We attribute this to the fact that the simultaneous production of carotenoids and lipids is advantageous for the cells to maintain proper structure, function and membrane homeostasis. We also highlight the need for further developments in carotenoids extraction methods to advance microbial bioprocess technology. In addition, we provide information on recent carotenoids extraction technology using two phase media, developed in an engineered strain of Rhodosporidium toruloides. The in depth understanding of this yeast would help future developments to increase the yield and extraction of carotenoids and lipid.

The strategies that High Risk Human Papillomavirus (HR-HPVs) have developed in differentiating epithelial cells to reach a DNA-synthesis competent state are basically related to the overexpression of the E6 and E7 oncoproteins and the cellular regulatory pathways that are targeted by them. These are basically related to hallmarks of cancer, and include sustaining proliferative signals, the evasion of growth suppression and immune destruction, replicative immortality, inflammation, invasion, metastasis and angiogenesis, as well as genome instability, resisting cell death and deregulation of cellular energetics. In this minireview, we summarize some of the biological activities of HVP16 E6 and E7 oncoproteins in cervical carcinogenesis. From this, it is evident that cervical cancer may represent an important model to understand the different oncogenic mechanisms and processes involved not only in this neoplasia but also in the development of other human malignancies. Likewise, the miRNAs and cancer stem cellrelated markers expression could serve as novel molecular targets for the diagnosis and treatment of HPV positive cervical cancer.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis resulting in cellular dysplasia and peripheral cytopenias. Research into MDS have found that both hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) in the nurturing niche of HSCs are genetically altered in MDS. In this review we present the existing views on the origin of cytogenetic abnormalities in HSC and MSC compartments in MDS. Based on the available data, we speculate that the origin of the chromosomal aberrations of the hematopoietic compartment occurs at the hematopoietic stem/ progenitor level. The genetic aberrations in MSC compartment appears to initiate independently from their hematopoietic counterparts. Whether the genetic events in both HSC and MSC compartments which lead to MDS occur simultaneously or at different time points in the disease development need to be determined in future.

Uromodulin (UMOD) gene mutation causes autosomal dominant Uromodulin-Associated Kidney Disease (UAKD), which in turn leads to end-stage renal disease. This is the first case report of a family with UAKD caused by a novel de novo mutation (E197X) in the UMOD gene. This case is a 28-year-old man with severely reduced kidney function [1]. No similar case was reported in his family history. This report highlights and reminds the importance of genetic screening in young patients involving kidney dysfunction, as the UAKD and some other kidney genetic diseases may be late-onset.

Objective: Neural tube defect (NTD) incidence could be effectively reduced by folic acid supplementation before and during pregnancy. We studied single nucleotide polymorphisms (SNPs) involved in folate metabolism to explore genetic susceptibility to NTD. We studied the association between 12 SNPs involved in 11 folate metabolism genes and NTDs.

Methods: We enrolled 76 children with NTD and 188 control children. We genotyped 12 folate metabolism SNPs including CBS-C699T, DHFR-c594+59del19, GSTO1-C428T, MTHFD-G1958A, MTHFR-C677T, MTHFR-A1298C, MTR-A2756G, MTRR-A66G, NFE2L2-ins1+C11108T, RFC1-G80A, TCN2-C776T, and TYMS-1494del6 using SNaPShot genotyping technology and confirmed by Sanger sequencing.

Results: One SNP, TYMS-1494del6, and one compound wide-type genotype of RFC1-G80A, MTHFR-A1298C and TCN2-C776T might decrease NTD risk, and three compound mutation genotypes of MTHFD-G1958A, MTHFRC677T, and MTR-A2756G; MTHFD-G1958A, MTR-A2756G, and RFC1-G80A; and RFC1-G80A, MTHFR-A1298C, and TCN2-C776T might increase NTD risk. The TT genotype of TYMS-1494del6 (P<0.001) and the AT+TT genotype of TYMS-1494del6 (P=0.009) were significant genotypes. Fourteen in 188 control babies carried the compound wide-type genotype of RFC1-G80A, MTHFR-A1298C, and TCN2-C776T, but none in 76 NTD babies (P=0.014). The ratios of the two compound mutants for MTHFD-G1958A, MTHFR-C677T, MTR-A2756G, and MTHFD-G1958A, RFC1-G80A, MTR-A2756G in NTD were higher than in control babies (P=0.021) and RFC1- G80A, MTHFR-A1298C, and TCN2-C776T (P=0.029).

Conclusions: The TT genotype of TYMS-1494del6 and the two wide-type genotypes of RFC1-G80A, MTHFRA1298C, and TCN2-C776T are protective in NTD. Three compound mutation genotypes of MTHFD-G1958A, MTHFR-C677T, MTR-A2756G, MTHFD-G1958A, MTR-A2756G, RFC1-G80A, and RFC1-G80A, MTHFR-A1298C, TCN2-C776T might increase susceptibility to NTD.

Background: Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (CRS+HIPEC) has been proposed as treatment for recurrent epithelial ovarian carcinoma. We evaluated the outcomes of CRS +HIPEC in recurrent epithelial ovarian cancers, in Indian patients.

Method: In this prospective non-randomized study between February 2013 and January 2015, 26 patients with advanced recurrent EOC, with no extra-abdominal disease treated with secondary CRS+HIPEC in a tertiary care cancer institution, Southern India, were analyzed. Belmonte® hyperthermia (HIPEC) pump with cisplatin 100 mg/m2, 41.5°C to 43°C for 90 minutes, in platinum sensitive cases and doxorubicin 15 mgs/m2 + cisplatin 75 mg/m2 in platinum resistant cases was used.

Result: Among twenty six patients 18 were upfront and 8 were post chemotherapy. Median peritoneal carcinomatosis Index was 9.5 (Range: 3-19). The extent of cytoreduction associated with longer hospital stay (p < 0.001), delayed gastrointestinal recovery (p=0.039), infections (p=0.036), and Acute Respiratory Distress Syndrome (p=0.041). Completeness of cytoreduction score CC0 achieved in 24 and CC1 in 2 patients. Bowel resection required in 34.6%. Diaphragm stripping was required in 30.7% with resection in 7.6%. Median hospital stay was 12 days (range: 10-42 days). No 30 days mortality. Bowel fistula happened in 7.6% cases requiring re-exploration, temporary stomas, and wound related complications in 26%. At median follow-up of eighteen months, 11.5% recurrences (both platinum resistant cases recurred in peritoneal cavity and one patient also in liver parenchyma) and one platinum sensitive patient recurred isolated in peritoneal cavity. One patient died at 5th month of follow up due to pulmonary embolism.

Conclusion: In our Indian study, secondary CRS+HIPEC are shown to be very promising in recurrent epithelial ovarian cancers patients with no extra-abdominal disease and good performance status. And can be done with acceptable morbidity, using dedicated HIPEC machine resulting in good peritoneal control of disease and disease free survival.

Pelvic organ prolapse (POP) is a multifactorial disease with a complex and largely unknown etiology and pathophysiology. Hypoestrogenemia may be one of the risk factors associated with POP. Recent studies suggest a potential role of estrogen and its receptors in the pathogenesis of POP. Here, we summarize current research regarding the relationship between estrogen and POP to establish a theoretical foundation for using estrogen in POP treatment. Estrogen plays an important role in collagen and elastin metabolism of connective tissues through down-regulating matrix metalloproteinases and increasing cystatin C expression. However, previous studies have shown contradictory data regarding estrogen receptor expression in patients with POP compared to non-POP controls. At this time, there is no conclusive evidence suggesting a causal role of estrogen in POP. Further welldesigned studies are necessary to illuminate both the molecular mechanisms of estrogen function and the role of estrogen in POP.

Objectives: Current study was the first to report a consanguineous Iranian pedigree with ABCD1 mutation.

Methods: Targeted molecular analysis was initially performed in three affected individuals in one family suspected to have X-ALD due to chronic progressive spasticity. Upon confirmation of genetic diagnosis, further neurologic and genetic evaluation of all family members was done.

Results: A mutation in ABCD1 was identified in 35 affected individuals (out 96 pedigree members). The c. 253dup, in exon 1, leads to a frame shift and a premature stop codon at amino acid position 194 (p.Arg85Profs*110). Surprisingly, affected individuals in our cohort show some variability in phenotype, including childhood cerebral ALD, adrenomyeloneuropathy, and addison-only disease phenotypes, expanding the phenotype of X-ALD with p.Arg85Profs*110.

Conclusion: This report characterizes the clinical spectrum of an expanded Iranian pedigree with X-ALD due to an ABCD1 mutation. Given a high frequency of carriers in this region, we expect the prevalence of X-ALD to be higher, underscoring the importance of genetic counseling through reliable identification of heterozygous as well as homozygote females in consanguineous communities.