The 21st century disaster: The COVID-19 epidemiology, risk factors and control.

The 21st century will be indelible in the world as ruin of the outbreak of COVID-19 was arose in Wuhan, China has now spread all over the world, up to August 2020. This study was based on the factors affecting the epidemiology of this virus in human societies of global concern. We studied the articles published in journals on various aspects of nCoVID19. The Wikipedia and WHO situation reports have also been searched out for related information. Outcomes were followed up until 2020. The COVID-19 is a virus with pandemic potential which may continue to cause regular infection in human. The pandemic outbreak of COVID-19 threatened public health across the globe in form of system as reflected in the shape of emergency. Approximately 21 million humans are infected and 759,400 have lost their lives till 2020 in all over the world. We have described epidemiological features, reservoirs, transmission, incubation period, rate of fatality, management including recent clinical chemotherapeutic approach and preventive measurements and masses which are at risk of COVID19. This virus causes viral pneumonia when it attacks on respiratory system and multiple failure which can leads to life threatening complications. It is believed to be zoonotic importance although it is not clear from which animal and how it is transmitted. Zoonotic transmission of COVID-19 has not yet known by science. The current study will help to establish a baseline for early effective control of this rapidly spreading severe viral illness. The available data on COVID-19 indicates that older males with comorbidities would have been more infected, which can result in severe respiratory complications. Implementation of preventive measurements, investigation of proper chemotherapeutics and detection of cross species transmission agents must be ensured.

Isolation, Characterization and Anticancer Activity of Two Bioactive Compounds from Arisaema flavum (Forssk.) Schott.

Medicinal plants play important role in the public health sector worldwide. Natural products from medicinal plants are sources of unlimited opportunities for new drug leads because of their unique chemical diversity. Researchers have focused on exploring herbal products as potential sources for the treatment of cancer, cardiac and infectious diseases. Arisaema flavum (Forssk.) is an important medicinal plant found in the northwest Himalayan regions of Pakistan. It is a poisonous plant and is used as a remedy against snake bites and scorpion stings. In this study, two bioactive compounds were isolated from Arisaema flavum (Forssk.) and their anticancer activity was evaluated against human breast cancer cell line MCF-7 using an MTT assay. The crude extract of Arisaema flavum (Forssk.) was subjected to fractionation using different organic solvents in increasing order of polarity. The fraction indicating maximum activity was then taken for isolation of bioactive compounds using various chromatographic and spectroscopic techniques such as column chromatography, thin-layer chromatography (TLC), gas chromatography−mass spectrometry (GC-MS), Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). Crude extract of Arisaema flavum (Forssk.), as well as various fractions extracted in different solvents such as n-hexane, chloroform and ethyl acetate, were tested against human breast cancer cell line MCF-7 using an MTT assay. The crude extract exhibited significant dose-dependent anticancer activity with a maximum activity of 78.6% at 500 µg/mL concentration. Two compounds, hexadecanoic acid ethyl ester with molecular formula C18H36O7 and molar mass 284 and 5-Oxo-19 propyl-docosanoic acid methyl ester with molecular formula C26H50O3 and molecular mass 410, were isolated from chloroform fraction. These compounds were tested against the MCF-7cell line for cytotoxic activity and exhibited a significant (p < 0.00l) decrease in cell numbers for MCF-7 cells with IC50 of 25 µM after 48 h of treatment. Results indicated that Arisaema flavum (Forssk.) possesses compounds with cytotoxic activity that can further be exploited to develop anticancer formulations.

Isolation, characterization, bacteriocin production and biological potential of Bifidobacteria of ruminants.

Bacteriocins exhibited a wide spectrum of antibacterial activity against different pathogens. The aim of current study was to characterize the bacteriocins produced by Bifidobacterium spp. isolated from ruminants. The Bifidobacterium isolates were identified as B. longum, B. pseudolongum, B. bifidum, B. thermophilum, B. boum, B. merycicum and B. ruminantium. Bacteriocins were found to be pH stable, heat resistant, highly diffusible, NaCl tolerant and resistant to UV radiations. SDS, EDTA and urea induced 14%, 21% and 24% bacteriocins activity loss. Modified MRS broth (1% tryptone, 1% yeast extract and 2% glucose) was found to be the best nutrient medium for optimal production of bacteriocins. Minimum inhibitory concentration (MIC) values varied from 300 µl/ml to 500 µl/ml and minimum bactericidal concentration (MBC) values ranged from 500 µl/ml to >500 µl/ml for E. coli and S. aureus respectively. The highest protein concentration (29.0248 mg/ml) was recorded for Bifidobacteria bacteriocin produced by B. longum. Tricine-Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) revealed that molecular weight of isolated bifidobacterial bacteriocins was in the range of 3.6 kDa-30 kDa. Current study indicated that bifidobacterial bacteriocins have considerable potential to be used as biopreservative.

Human papillomavirus, tobacco, and poor oral hygiene can act synergetically, modulate the expression of the nuclear factor kappa B signaling pathway for the development and progression of head and neck cancer in the Pakistani population.

BACKGROUND: Head and neck cancers (HNCs) are a heterogeneous group of tumors that progress owing to varied enviromental and genetic risk factors. Viral infections are threatening and adept at altering the expression of cellular transcription factors such as nuclear factor kappa B (NF-κB) and deregulation of other cellular proteins like NF kappa B inhibitor alpha (IκBα). The present study was conducted to detect high-risk genotypes of human papillomavirus (HPV) and protein expression of NF-κB signaling pathway in HNC patients with HPV infection. METHODS: For HPV detection, genomic DNA from 152 HNC tumors was extracted formalin-fixed paraffin-embedded tissue DNA kit. For genotyping, polymerase chain reaction (PCR) using a general primer, HPV type-specific primers and agarose gel electrophoresis were performed. Immunohistochemistry (IHC) was also performed on 4-µm thick tissue sections using HPV E6 monoclonal antibody. Protein expression analysis of NF-κB signaling pathway including p50, p65, and IκBα was performed using IHC. RESULTS: PCR analysis showed that 24.3% (37/152) of HNC cases were HPV positive. Among HPV positive, 86.5% (32/37) were tobacco users, while among HPV negative, 66.9% (77/115) were tobacco users. A significant association of HPV positivity and tobacco user was observed by univariate analysis [ P   <  0.01; odds ratio (OR): 0.310, 95% confidence interval (CI): 0.110 to 0.870]. More HPV positive patients were with poor oral hygiene (78.3%) when compared with patients with good oral hygiene (21.6%) [ P  < 0.03, OR: 2.440, 95% CI: 1.650 to 3.600]. The results of the logistic regression analysis showed that age, tobacco use and oral hygiene are significant predictors ( P  < 0.02). PCR and IHC staining results confirmed that HPV16 was predominant among HNC cases (64.8%) when compared with HPV18 (35.2%). Expression of NF-κB proteins (p50, p65, and IκBα inhibitor) were also observed in HPV and non-HPV infected HNC tissues. IHC expression of p50, and p65 showed nuclear staining, while IκBα inhibitor showed cytoplasmic staining. Protein expression in HPV cases was higher as compared to HPV naive cases ( P  < 0.05). CONCLUSIONS: From the study, it can be established that the use of tobacco, oral hygiene, and HPV infection may be synergistically involved in modulating the expression of NF-κB signaling pathway for the development and progression of HNC in the Pakistani population.

Therapeutic Potential of Selected Medicinal Plant Extracts against Multi-Drug Resistant Salmonella enterica serovar Typhi.

Salmonella enterica serovar Typhi is Gram negative, rod shaped, facultative anaerobic bacterium, belongs to enterobacteriaceae family that causes typhoid fever in humans. This bacterium has become a super bug due to acquisition of multi drug resistance. Bacteria is transmitted through food and water contaminated with human feaces. Present study reports the screening of Adhatoda vasica, Amaranthus hybridus and Aloe barbadensis and their evaluation against multi-drug resistant Salmonella enterica serovar Typhi. Qualitative analysis of ten phytochemicals was conducted using chemical method and Gas Chromatography-Mass Spectrometry (GCMS). Antibacterial activity of plants was carried out by agar well diffusion method on Mueller Hinton agar. Total tannins, total alkaloids and total flavonoids of different parts of three plants were estimated through spectrophotometer. Total tannins content in different parts of plants was present in the given order Amaranthus hybridus leaf > Aloe barbadensis leaf > Adhatoda vasica leaf > Adhatoda vasica flower > Adhatoda vasica stem. Whereas, the order of total flavonoid concentration was Amaranthus hybridus leaf > Aloe barbadensis leaf > Adhatoda vasica leaf > Amaranthus hybridus seed. Total alkaloids have order, Adhatoda vasica leaf > Amaranthus hybridus leaf > Adhatoda vasica flower > Amaranthus hybridus seed > Aloe barbadensis leaf. Results of phytochemical analysis suggested that plants have strong profile of antioxidants, total phenolic contents and various enzymes proposing them best alternate to cure bacterial infections. GC-MS analysis further confirmed stronger phytochemical profile that can be utilized as antagonists to Salmonella enterica serovar Typhi.

Fungicidal synergistic effect of biogenically synthesized zinc oxide and copper oxide nanoparticles against Alternaria citri causing citrus black rot disease.

Citrus black rot disease being caused by Alternaria citri is a major disease of citrus plants with 30-35% economic loss annually. Fungicides had not been effective in the control of this disease during last few decades. In the present study, antifungal role of green synthesized zinc oxide (ZnO) and copper oxide (CuO) nanoparticles (NPs) were studied against Alternaria citri. Alternaria citri was isolated from disease fruits samples and was identified by staining with lacto phenol cotton blue. Furthermore, CuO and ZnO NPs were synthesized by utilizing the lemon peels extract as the reducing and capping agent. Nanoparticles were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM) techniques. From the XRD data, the calculated size of CuO NPs was to be 18 nm and ZnO NPs was16.8 nm using Scherrer equation. The SEM analyses revealed the surface morphology of all the metal oxide NPs synthesized were rounded, elongated and or spherical in the shape. The zone of inhibition was observed to be 50 ± 0.5 mm by CuO NPs, followed by 51.5 ± 0.5 mm by ZnO NPs and maximum zone of antifungal inhibition was observed to be 53 ± 0.6 mm by mix metal oxide NPs. The results of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the synthesized nanoparticles showed that at the certain concentrations (80 mg ml-1), these NPs were capable of inhibiting the fungal growth, whereas above that specified concentrations (100 mg ml-1), NPs completely inhibited the fungal growth. Based on these findings, the green synthesized NPs can be used as alternative to fungicide in order to control the citrus black rot disease.

Therapeutic prospects of endophytic Bacillus species from Berberis lycium against oxidative stress and microbial pathogens.

Endophytes are microorganisms residing within plant tissues. Bacterial endophytes are important sources for production of pharmaceutically important metabolites. Berberis lycium is an important medicinal plant and there exist no report regarding isolation and determination of bioactive potential of its bacterial endophytes. Therefore the present study was aimed to isolate and identify bacterial endophytes from Berberis lycium. The study resulted in isolation of 20 strains of bacterial endophytes. Based on their antibacterial activity three strains were identified as Bacillus cereus (LBL6), Bacillus thuringiensis (SBL3) and Bacillus anthracis (SBL4) on basis of 16SrRNA gene using universal primers. Crude ethyl acetate extracts of LBL6, SBL3 and SBL4 were further evaluated for antioxidant and antifungal activities. Moderate antioxidant activity (56 %) at a concentration of 1000 µg/mL was observed for LBL6 followed by 45 and 43 % activity by SBL4 and SBL3 respectively. Significant antifungal activity was observed against Aspergillus niger (60 %) and Aspergillus flavus (56 %) at concentration of 4 mg/mL of SBL3 and SBL4 respectively. GCMS analysis of extract (LBL6) exhibited presence of 12 bioactive secondary metabolites corresponding to antimicrobial, antifungal, antioxidant, antitumor and anticancer activities. In conclusion, present study highlighted the importance of Berberis lycium to host diverse bacterial endophytes of pharmaceutical importance.

Bacillus species; a potential source of anti-SARS-CoV-2 main protease inhibitors.

The COVID-19 being a preconized global pandemic by the World Health Organization needs persuasive immediate research for possible medications. The present study was carried out with a specific aim to computationally evaluate and identify compounds derived from Bacillus species as the plausible inhibitors against 3-chymotrypsin-like main protease (3CLpro) or main protease (MPro), which is a key enzyme in the life-cycle of coronavirus. The compounds were isolated from the crude extracts of Bacillus species. Among the isolated compounds, novel inhibitory leads were identified using in silico techniques. Molecular docking revealed that stigmasterol (-8.3 kcal/mol), chondrillasterol (-7.9 kcal/mol) and hexadecnoic acid (-6.9 kcal/mol)) among others bind in the substrate-binding pocket and also interacted with the catalytic dyad of the 3-CLpro. Further evaluation using 50 ns molecular dynamic simulation and MMPB-GBSA indicated that among the top three docking hits, hexadecanoic acid was found to be the most promising anti-COVID-19 lead against the main protease. Hexadecanoic acid might serve as a potent anti-SARS-CoV-2 compound to combat COVID-19, however, in vitro and in vivo validation and optimization is needed.Communicated by Ramaswamy H. Sarma.

NF-κB1 Intronic Region Polymorphisms as Risk Factor for Head and Neck Cancer in HPV-Infected Population from Pakistan.

BACKGROUND: Head and neck cancer (HNC) develops due to a number of risk factors, including infection of Human Papillomavirus (HPV). The genetic predisposition also plays an important role in deregulating different signaling pathways including the NF-KB pathway. Certain polymorphisms are reported to affect the NF-kB pathway genes. OBJECTIVES: The present research was conducted to study the association of HPV with NF-KB1 (p50) gene polymorphisms in HNC patients of the Pakistani population. METHODS: Genomic DNA from HNC tumors samples was extracted using the Exgene SV DNA extraction Kit. Allele-specific PCR and direct sequencing were done for analysis of NF-κB1 SNPs 94ins/del (rs28362491), rs1598858 and rs4648068. RESULTS: The genotypes AGrs1598858, AGrs4648068 and GGrs4648068 were associated with significantly increased risk of head and neck cancer in studied population. Furthermore the HNC cases with genotypes AGrs1598858 and GGrs4648068 displayed growing risk of HPV related cancers. Promotor region SNP 94ins/del (rs28362491) was not detected in studied population. Tobacco use, lymph nodes involvement and poorly differentiated tumors were positively associated with HPV induced cancers. CONCLUSION: It is the first comprehensive study from Pakistan, to evaluate the polymorphic variants of NF-κB1. Genotypes AGrs4648068, GGrs4648068, and AGrs1598858 of NF-κB1 gene are associated with increased risk of head and neck cancers in the studied HPV infected Pakistani population. It can be concluded that HPV infection, involvement of lymph nodes and tobacco use can act synergetic and add up in modulating HPV induced HNC with intronic SNPs of NF-κB1 gene in Pakistani population.

Pharmacological properties of biogenically synthesized silver nanoparticles using endophyte Bacillus cereus extract of Berberis lyceum against oxidative stress and pathogenic multidrug-resistant bacteria.

The emergence of multidrug resistance in pathogenic bacteria limits the utilization of available antibiotics. The development of alternate options to treat infectious diseases is the need of the day.The present study was aimed to synthesize, characterize and evaluate the bioactive properties of silver nanoparticles. Endophytic bacterium Bacillus cereus (MT193718) isolated from Berberis lycium was used to synthesize biocompatible silver nanoparticles. Antibacterial properties of AgNPs were evaluated against clinically isolated multidrug-resistant strains of Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae. AgNPs indicated significant antibacterial activity against S. aureus and K. pneumoniae fwith a zone of inhibition of 17 and 18 mm at a concentration of 1000 µg/ mL with minimum inhibitory concentration of 15.6 and 62.5 µg/mL respectively. Significant antioxidant activity with an IC50 value of 9.5 µg/mL was recorded. Biosynthesized AgNPs were found compatible with red blood cells at a concentration of 31.5 µg/ml with no clumping of erythrocytes. The study suggested that AgNPs synthesized by the endophytic bacterium Bacillus cereus are biologically active and can be used as antioxidant and antibacterial agents against drug-resistant bacteria.

Environmental impacts, water footprint and cumulative energy demand of match industry in Pakistan.

A comprehensive life cycle assessment (LCA) was conducted for the matchsticks industry in the Khyber Pakhtunkhwa province of Pakistan to quantify environmental footprint, water footprint, cumulative energy use, and to identify improvement opportunities in the matchsticks manufacturing process. One carton of matchsticks was used as reference unit for this study. Foreground data was collected from the matchsticks industry through questionnaire surveys, personal meetings, and field measurements. The collected data was transformed into potential environmental impacts through the Centre for Environment Studies (CML) 2000 v.2.05 method present by default in the SimaPro v.9.1 software. Water footprint was calculated using methodology developed by Hoekstra et al., 2012 (water scarcity index) V1.02 and cumulative energy demand by SimaPro v.9.1 software. The results showed that transport of primary material (wood logs), sawn wood for matchsticks, red phosphorous, acrylic varnish, and kerosene fuel oil contributed to the overall environmental impacts. Transport of primary materials and sawn timber for matchsticks contributed significantly to abiotic depletion, global warming, eutrophication potential, ozone depletion, corrosion, human toxicity, and aquatic ecotoxicity effects. The total water footprint for manufacturing one carton of matchsticks was 0.265332 m3, whereas the total cumulative energy demand was 715.860 Mega Joules (MJ), mainly sourced from non-renewable fossil fuels (708.979 MJ). Scenario analysis was also conducted for 20% and 30% reduction in the primary material distance covered by trucks and revealed that reducing direct material transport distances could diminish environmental impacts and energy consumption. Therefore, environmental footprint could be minimized through diverting matchsticks industries freight from indigenous routes to high mobility highways and by promoting industrial forestry close to industrial zones in Pakistan. Many industries did not have emissions control systems, exceeding the permissible limit for emissions established by the National Environmental Quality Standards (NEQS) of Pakistan. Thus, installation of emissions control system could also diminish emissions from match industry in Pakistan.

OGG1 Mutations and Risk of Female Breast Cancer: Meta-Analysis and Experimental Data.

In first part of this study association between OGG1 polymorphisms and breast cancer susceptibility was explored by meta-analysis. Second part of the study involved 925 subjects, used for mutational analysis of OGG1 gene using PCR-SSCP and sequencing. Fifteen mutations were observed, which included five intronic mutations, four splice site mutations, two 3'UTR mutations, three missense mutations, and a nonsense mutation. Significantly (p < 0.001) increased (~29 fold) breast cancer risk was associated with a splice site variant g.9800972T>G and 3'UTR variant g.9798848G>A. Among intronic mutations, highest (~15 fold) increase in breast cancer risk was associated with g.9793680G>A (p < 0.009). Similarly ~14-fold increased risk was associated with Val159Gly (p < 0.01), ~17-fold with Gly221Arg (p < 0.005), and ~18-fold with Ser326Cys (p < 0.004) in breast cancer patients compared with controls, whereas analysis of nonsense mutation showed that ~13-fold (p < 0.01) increased breast cancer risk was associated with Trp375STOP in patients compared to controls. In conclusion, a significant association was observed between OGG1 germ line mutations and breast cancer risk. These findings provide evidence that OGG1 may prove to be a good candidate of better diagnosis, treatment, and prevention of breast cancer.

Germline variations of apurinic/apyrimidinic endonuclease 1 (APEX1) detected in female breast cancer patients.

Apurinic/apyrimidinic endonuclease 1 (APEX1) is a multifunctional protein which plays a central role in the BER pathway. APEX1 gene being highly polymorphic in cancer patients and has been indicated to have a contributive role in Apurinic/apyrimidinic (AP) site accumulation in DNA and consequently an increased risk of cancer development. In this case-control study, all exons of the APEX1 gene and its exon/intron boundaries were amplified in 530 breast cancer patients and 395 matched healthy controls and then analyzed by single-stranded conformational polymorphism followed by sequencing. Sequence analysis revealed fourteen heterozygous mutations, seven 5'UTR, one 3 'UTR, two intronic and four missense. Among identified mutations one 5'UTR (rs41561214), one 3'UTR (rs17112002) and one missense mutation (Ser129Arg, Mahjabeen et al., 2013) had already been reported while the remaining eleven mutations. Six novel mutations (g.20923366T>G, g.20923435G>A, g.20923462G>A, g.20923516G>A, 20923539G>A, g.20923529C>T) were observed in 5'UTR region, two (g.20923585T>G, g.20923589T>G) in intron1 and three missense (Glu101Lys, Ala121Pro, Ser123Trp) in exon 4. Frequencues of 5'UTR mutations; g.20923366T>G, g.20923435G>A and 3'UTR (rs17112002) werecalculated as 0.13, 0.1 and 0.1 respectively. Whereas, the frequency of missense mutations Glu101Lys, Ser123Trp and Ser129Arg was calculated as 0.05. A significant association was observed between APEX1 mutations and increased breast cancer by ~9 fold (OR=8.68, 95%CI=2.64 to 28.5) with g.20923435G>A (5'UTR) , ~13 fold (OR= 12.6, 95%CI=3.01 to 53.0) with g.20923539G>A (5'UTR) and~5 fold increase with three missense mutations [Glu101Lys (OR=4.82, 95%CI=1.97 to 11.80), Ser123Trp (OR=4.62, 95%CI=1.7 to 12.19), Ser129Arg (OR=4.86, 95%CI=1.43 to 16.53)]. The incidence of observed mutations was found higher in patients with family history and with early menopause. In conclusion, our study demonstrates a significant association between germ line APEX1 mutations and breast cancer patients in the Pakistani population.

Retinoblastoma (RB1) pocket domain mutations and promoter hyper-methylation in head and neck cancer.

BACKGROUND: The RB1 gene plays a pivotal role in cell cycle regulation. In this case-control study we searched for alterations in the RB1 pocket domain and its promoter region in head and neck cancer (HNC) patients in the Pakistani population. METHODS: For germline mutation analyses, 380 blood samples from HNC patients and 350 blood samples from control individuals were included. Polymerase chain reaction (PCR) and single strand conformational polymorphism (SSCP) assays, followed by sequence analyses, were used for the RB1 pocket domain mutation screens. For the RB1 promoter methylation screens, 72 HNC tumor samples along with adjacent uninvolved tissues were tested using a methylation-specific polymerase chain reaction (MSP) assay. RESULTS: RB1 (pocket domain and spacer region) sequence analysis revealed one frameshift and seven non-synonymous missense mutations. The frequency of missense mutations in exon 14, i.e., g76474C > T, g76475G > C and g76476A > G, resulting in Arg455Ser, was found to be highest (0.10). Missense mutations g76467G > C (exon14), g76468T > C (exon14), g77041A > T and g77043A > T (exon 16), when analyzed via Alamut biosoftware version 2.0, were found to be present in highly conserved amino acids with Align GVGD scores C15 (GV: 0.00-GD: 21.82), C65 (GV: 0.00-GD: 83.33) and C65 (GV: 0.00-GD: 98.69), respectively. These missense mutations were found to be deleterious by SIFT score: 0.00 (median 3.64). RB1 promoter methylation analysis revealed that 16% of its cytosines (3% in CpG) were methylated in the HNC tumor samples. CONCLUSION: Our findings indicate that both genetic and epigenetic RB1 changes may contribute to the pathogenesis of HNC in the Pakistani population.

Genetic and expressional variations of APEX1 are associated with increased risk of head and neck cancer.

The aetiology of head and neck cancer (HNC) has been shown to be associated with genetic and certain environmental factors that produce DNA damage. Base excision repair (BER) genes are responsible for repair of DNA damage caused by reactive oxygen species and other electrophiles and therefore are good candidate susceptibility genes for HNC. Apurinic/apyrimidinic endonuclease-1 (APEX1) proteins have important functions in the BER pathway. In this case-control study, all exons of the APEX1 gene and its exon/intron boundaries were amplified in 300 HNC cases and 300 matched healthy controls and then analysed by single-stranded conformational polymorphism. Amplified products showing altered mobility patterns were sequenced and analysed. To confirm our observations, we examined APEX1 expression at mRNA level on 50 head and neck squamous cell carcinoma (HNSCC) and 50 normal control samples by quantitative real-time polymerase chain reaction. At germ line level, three novel mutations (13T > G, Ser129Arg and Val131Gly) of APEX1 were observed. The homozygous and heterozygous genotypes of APEX1 13T > G, Ser129Arg and Val131Gly appear to be significantly involved in the development of HNC. In the case of expressional level, APEX1 mRNA expression was positively correlated with tumour size, clinical stage and positive lymph node metastasis. Statistical analysis showed a significantly higher APEX1 mRNA level in HNC tumour tissue than in control samples. Our study demonstrated that APEX1 mutations and deregulation of APEX1 are associated with increased risk of HNC in the Pakistani population.

Mutational spectrum of Gelsolin and its down regulation is associated with breast cancer.

Cytoskeletal rearrangement occurs in variety of cellular processes and involves a wide spectrum of proteins. Gelsolin super family proteins control actin organization by severing and capping filament ends and nucleating actin assembly. Gelsolin is the founding member of this family and plays important role in pathogenesis of human neoplasia. This study aimed to investigate the germline mutations and expressional profile of Gelsolin in human breast cancer tissues. For germ line screening PCR-SSCP technique was used while expression was analyzed through quantitative real time PCR. Different types of mutations were observed in Gelsolin coding regions on exons 4, 10, 11, 14 and 15. These mutations include 3 missense nonsynonymous substitution mutations, 2 deletions, 1 insertion and 1 synonymous substitution mutation. Gelsolin transcript level was found significantly lower in breast tumor tissues compared to control samples (p=0.03). Low level of Gelsolin was found in metastatic patients (p=0.002) and patients who died from breast cancer (P=0.03) compared to disease free patients at final follow up. This study shows that level of Gelsolin is down regulated in breast cancer tissues and is linked with metastasis development and death in patients. It is concluded that genetic changes in coding regions of Gelsolin can potentially contribute to genetic instability. These genetic variations and expressional correlation with patient survival may prove to be of significant importance.

Genetic variations in XRCC1 gene in sporadic head and neck cancer (HNC) patients.

DNA repair gene polymorphisms have been implicated as susceptibility factors in cancer development. It is possible that DNA repair polymorphisms may also influence the risk of gene mutation. Polymorphisms in the DNA repair gene XRCC1 have been indicated to have a contributive role in DNA adduct formation and an increased risk of cancer development. 300 head and neck cancer patients and 150 controls were included in this study. PCR-single-strand conformation polymorphism and DNA sequencing were used to analyze the whole exonic region of XRCC1 in head and neck cancer patients. Sequence analysis revealed two missense and two silent mutations in our study. Frequency of silent mutations; Pro206Pro (rs915927) and Gln632Gln (rs3547) was calculated as 0.16 (16 %) and 0.30 (30 %) respectively. Whereas, the frequency of missense mutations; Arg399Gln (rs25487) and Tyr576Asn (rs2307177) was calculated as 0.27 (27 %) and 0.28 (28 %) respectively. In our study, incidence of these mutations was found higher in larynx cancer (p < 0.005) as compared to oral cavity and pharynx cancer. Our finding suggests that the polymorphic XRCC1 gene may contribute to risk of developing head and neck cancer. To our knowledge, this is the first report that XRCC1 is associated with increased risk of head and neck cancer in a Pakistani population.

Significance of cyclin D1 polymorphisms in patients with head and neck cancer.

Cyclin D1 plays a key role in cell cycle control, particularly in the transition from G1 to S phase, regulated by cyclin-dependent kinases. The objective of the present study was to screen the cyclin D1 gene (CCND1) for polymorphisms in patients with head and neck cancer (HNC). Genomic DNA was isolated from blood samples of 380 HNC patients and 350 controls. In a hospital-based case-control study using the PCR-SSCP technique we found 3 novel germline mutations: g3578C>A, g3475G>C and g3383delA. The commonly reported guanine to adenine polymorphisms in exon 4 g7656G>A (rs9344) and g10861C>A (rs7177) in 3'UTR of CCND1 were also observed. The calculated frequencies of the g7656G>A (rs9344) polymorphism in GG, GA and AA genotypes were 27.3%, 38.6%, and 33.9% in HNC cases, and 44.2%, 29.4%, and 26.2% in normal healthy controls, respectively. Adjusted by age (in years), sex and smoking status, multivariate logistic regression analysis showed that the AA and GA genotypes were associated with a significantly increased risk (OR 1.34, 95% CI 1.03-1.64, p=0.028) for HNC. The CCND1 AA genotype variant was associated with an increased risk in individuals who were <40 years old (OR 1.45, 95% CI 1.02-2.08, p=0.04). In conclusion, it is suggested that the CCND1 G/A polymorphism is associated with the early onset of HNC and may contribute to HNC susceptibility in a Pakistani population.

Novel germline CDK4 mutations in patients with head and neck cancer.

BACKGROUND: Cyclin-dependent kinase 4 (CDK4) together with its regulatory subunit cyclin D1, governs cell cycle progression through G1 phase. Cyclin-dependent kinase inhibitors, including p16INK4A in turn regulate CDK4. In particular, deregulation of the p16/CDK4/cyclin D1 complex has been established in a variety of human tumors including gliomas, sarcomas, melanoma, breast and colorectal cancer. However, changes in CDK4 have rarely been observed. METHOD: In this study we used a combination of PCR-SSCP and direct sequencing for mutational screening of CDK4. DNA was isolated from peripheral blood leukocyte of patients with squamous cell carcinoma of head and neck, for screening germline mutations in coding regions of CDK4. RESULTS: Variations observed in exon 2 and 5 were three missense mutations, g5051G > C (Ser52Thr), g5095G > C (Glu67Gln), g5906C > A, g5907C > G (Pro194Ser) and novel frame shift mutations g7321_23delTGA, g7121_7122insG, g7143delG in exon 7 and 3'UTR respectively. CONCLUSION: In conclusion, two novel mutations were found in N terminal domain which indicates that CDK4 mutation may play a major role in the development and progression of squamous cell carcinoma of head and neck.

Novel mutations of OGG1 base excision repair pathway gene in laryngeal cancer patients.

OGG1 (The human 8-oxoguanine glycosylase 1) is the primary enzyme in BER (base excision repair) pathway, responsible for the excision of 7, 8-dihydro-8-oxoguanine (8-oxoG), a mutagenic base byproduct that occurs as a result of exposure to reactive oxygen species. OGG1 gene is highly polymorphic among humans and is mutated in cancer cells. In this case control study, all exons of OGG1 gene and its exon/intron boundaries were amplified in 210 laryngeal cancer cases and 210 matched controls and then analyzed by single stranded conformational polymorphism. Amplified products showing altered mobility patterns were sequenced and analyzed. Two silent (Gln718Gln, His699-700His) and three missense (Ala597, Thr608-610Pro and Glu707Lys) mutations were observed in exon 2. In addition to this one missense mutation (1578G > A) was also observed in 3'UTR region. We found a significant association between OGG1 mutations and laryngeal cancer and observed that His699-His700, silent mutation exhibited an enhanced risk of ~9.0 folds (OR = 9.07, 95 % CI = 4.73-17.39) and 1578G > A, missense mutation ~0.4 folds (OR = 0.37, 95 % CI = 0.15-0.90). Furthermore, a positive association of OGG1 mutations with smoking was observed in laryngeal cancer cases when compared to controls. Heavy smokers have higher incidence of OGG1 mutations when compared to light smokers in present study. Our results demonstrate that OGG1 mutations are associated with an increased risk of laryngeal cancer. OGG1 mutations were found to accumulate more of 8-OHdG in smokers, which may serve as a biomarker for early diagnosis of laryngeal cancer.