Synthesis of Silver Nanoparticles from Ganoderma Species and Their Activity against Multi Drug Resistant Pathogens.

The widespread and indiscriminate use of broad-spectrum antibiotics leads to microbial resistance, which causes major problems in the treatment of infectious diseases. However, advances in nanotechnology using mushrooms have opened up new domains for the synthesis and use of nanoparticles against multidrug-resistant pathogens. Mushooms have recently attracted attention and are exploited for food and medicinal purposes. The current study focuses on the molecular identification, characterization of biologically synthesized silver nanoparticles by X-ray diffraction (XRD) spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), UV-Vis spectroscopy and scanning electron microscopy (SEM) and antibacterial analysis of extract and silver nanoparticles (AgNPs) synthesis from Ganoderma resinaceum against multidrug resistant microbes. Accurate identification of mushrooms is key in utilizing them for the benefit of humans. However, morphological identification of mushrooms is time consuming, tedious and may be prone to error. Molecular techniques are quick and reliable tools that are useful in mushroom taxonomy. Blast results showed that G. resinaceum (GU451247) obtained from Pakistan was 97 % same to the recognized G. resinaceum (GU451247) obtained from China as well as G. resinaceum (GU451247) obtained from India. The antimicrobial potential of mushroom composite and AgNPs showed high efficacy against pathogenic Staphylococcus aureus (ZOI 23 mm) K. pneumonia (ZOI 20 mm), Pseudomonas aeruginosa (ZOI 24 mm) and E. fecalis and A. baumannii (ZOI 10 mm), and multidrug resistant (MDR) A. baumannii (ZOI 24 mm). XRD evaluation revealed the crystalline composition of synthesized NPs with diameter of 45 nm. UV-Vis spectroscopy obsorption peaked of 589 nm confirmed the presence of AgNPs. SEM results showed the cubic morphology of AgNPs. The FTIR analysis of NPs obtained from G. resinaceum containing C=O as well as (O=C-H) stretching revealed presence of hydrogen, carbonyl and amide groups. The synthesized extract and AgNPs showed promising minimum inhibitory concentration (MIC) at 2 mg concentration against the MDR strains. AgNPs are observed to be efficient as they need less quantities to prevent bacterial growth. In the view of challenges for developing antimicrobial NPs of variable shape and size by various other methods, tuning nanoparticles synthesized via mushrooms can be a wonderful approach to resolve existing hurdles.

Molecular Profiling, Characterization and Antimicrobial Efficacy of Silver Nanoparticles Synthesized from Calvatia gigantea and Mycena leaiana against Multidrug-Resistant Pathogens.

The use of natural products isolated from mushrooms against infection, cancer diseases and other oxidative-stress-related diseases is one of the cornerstones of modern medicine. Therefore, we tried to establish a combination of medicinal mushrooms and nanotechnology possibly with the field of medicine for the development of antibacterial agents against these MDR strains. The aim of the research was to understand the molecular identification, characterization and antibacterial action of Calvatia gigantea and Mycena leaiana. The identification of fruiting body species via morpho-anatomical and molecular methods was necessary to analyze the genetic variability and phylogenetic relationships of mushrooms. Phylogenetic analysis revealed that Calvatia from Hunza, Pakistan, exhibited 98% resemblance to the previously discovered Langermannia gigantean (DQ112623) and L. gigantean (LN714562) from northern Europe, and Mycena (Pakistan) showed a 97% similarity to M. leaiana (MF686520) and M. leaiana (MW448623) from the USA. UV-vis, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) were used for AgNPs' characterization. The UV-vis absorption peak of 500-600 nm indicates the AgNPs' presence. XRD results determined Calvatia gigantea AgNPs were nanocrystals and Mycena leaiana seems to be amorphous. In addition, SEM results showed the cubic morphology of C. gigantea with a diameter of 65 nm, and the FTIR spectra of fruiting body revealed the presence of functional groups-carboxyl, nitro, and hydroxyl-in AgNPs, which catalyzed the reduction of Ag+ to Ag0. Further antibacterial activity of mushrooms against MDR strains was determined via agar well diffusion assay, and Minimum Inhibitory Concentration (MIC) was estimated by qualitative experimentation using the broth dilution method. All experiments were conducted in triplicate. The results showed that the mushroom AgNPs, along with their synergy and nano-composites (with the exception of Ethyl-acetate), were shown to have zones of inhibition from 4 mm to 29 mm against multidrug-resistant pathogens such as Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Proteus mirabilis, Enterobacter cloacae and Escherichia coli. The mushroom composites were active against most of the tested microorganisms whilst the lowest MIC value (10-40 mg/mL) was recorded against MDR strains. Hence, the present study suggested the possibility of employing compounds present in mushrooms for the development of new antibacterial agents, as well as efflux pump inhibitors.

Antimicrobial activity of AgNO3 nanoparticles synthesized using Valeriana wallichii against ESKAPE pathogens.

The emergence of multidrug-resistant ESKAPE infections has emerged as a serious public health threat. Nosocomial infections are most often caused by ESKAPE bacteria. To combat multidrug-resistant ESKAPE, the research team used Valeriana Wallichii extracts and nanoparticles. The well diffusion technique was used to test antimicrobial activity on Muller Hinton agar medium. The FTIR, SEM and XRD techniques were used to characterize the nanoparticles synthesized in an environmentally benign manner. Both NPs performed better than extracts made with methanol and water in this investigation. The smallest zones of inhibition were shown against A. baumannii and Enterobacter cloacae, whereas the largest zones of inhibition were seen against E. faecium. However, NPs synthesized from shoot extracts exhibited remarkable effects against all MDR ESKAPE infections, with zones of inhibition of 23, 20, 12, 18, 22 and 14mm, respectively. Although E. faecium. had the largest inhibitory zone in both methanolic root and shoot extracts (19mm and 22mm, respectively), K. pneumonia and E. cloacae had the smallest zones when tested with these solvents. Water-based extracts inactivated multidrug-resistant bacteria. Our research show that extracts and nanoparticles have stronger antibacterial efficiency because biologically active substances including Terpenoids, Alkaloids, Phenol and Pholobutannins affect people and microbe.

Antimicrobial susceptibility pattern and genotypic characterisation of quinolone and ceftriaxone resistant genes among Salmonella typhi from various regions of Pakistan.

OBJECTIVE: To determine the current antibiotic resistance patterns and identification of quinolone and ceftriaxone resistant genes among Salmonella enterica subspecies serovar Typhi. METHODS: The prospective study was conducted from September 2018 to March 2019 and comprised samples collected from major hospitals and laboratories in Karachi, Quetta, Lahore, Kharia, Rawalpindi, Islamabad and Peshawar after approval from the institutional ethics review board of Hazara University, Mansehra, Pakistan. Antimicrobial susceptibility of isolates collected from the health facilities was checked using the Kirby Bauer disc diffusion method in line with the Clinical and Laboratory Standards Institute guidelines at the Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan. All isolates were subjected for identification of genes responsible for quinolone and ceftriaxone resistance using polymerase chain reaction followed by gel-electrophoresis. RESULTS: Among the 96 isolates, phenotypically, ceftriaxone was found resistant in 31(32.29%) and ciprofloxacin in 95(99%). Genotypically, blaCTX-M-15 (beta lactamase, CTX as its acronym, -M from Munich) gene for ceftriaxone resistance was found in all phenotypically resistant 31(32.29%) isolates, while QnrS (Quinolone resistance, S group), GyrA (DNA gyrase subunit A), and GyrB (DNA gyrase subunit B) genes responsible for ciprofloxacin resistance were found in different frequencies (percentages given in table 2). CONCLUSIONS: The spread of extensively drug-resistant Salmonella enterica subspecies serovar Typhi strain to many big cities calls for urgent preventive measures.

Comparative Effect of Fertilization Practices on Soil Microbial Diversity and Activity: An Overview.

Continuously increasing human population demands increased food production, which needs greater fertilizer's input in agricultural lands to enhance crop yield. In this respect, different fertilization practices gained acceptance among farmers. We reviewed effect of three main fertilization practices (Conventional-, Organic-, and Bio-fertilization) on soil microbial diversity, activity, and community composition. Studies reported that over application of inorganic fertilizers decline soil pH, change soil osmolarity, cause soil degradation, disturb taxonomic diversity and metabolism of soil microbes and cause accumulation of extra nutrients into the soil such as phosphorous (P) accumulation. On the contrary, organic fertilizers increase organic carbon (OC) input in the soil, which strongly encourage growth of heterotrophic microbes. Organic fertilizer vermicompost application provides readily available nutrients to both plants as well as microbes and encourage overall microbial number in the soil. Most recently, role of beneficial bacteria in long-term sustainable agriculture attracted attention of scientists towards their use as biofertilizer in the soil. Studies documented favorable effect of biofertilization on microbial Shannon, Chao and ACE diversity indices in the soil. It is concluded from intensive review of literature that all the three fertilization practices have their own way to benefit the soil with nutrients, but biofertilization provides long-term sustainability to crop lands. When it is used in integration with organic fertilizers, it makes the soil best for microbial growth and activity and increase microbial diversity, providing nutrients to soil for a longer time, thus improving crop productivity.

An overview of self-medication: A major cause of antibiotic resistance and a threat to global public health.

Self-medication is the use of medicines by people on the basis of their own experience without consulting a doctor. People use medicines for pain management or to cure a disease and sometime this may be unnecessary. There are a lot of public and professional health concerns about misuse of medicines and globally physicians agree upon this rising issue that leads to antibiotic resistance. In developing countries, medicines without prescription are easily available which results in many adverse outcomes, especially bacterial resistance. Insufficient healthcare services and socioeconomic factors result in increased proportion of self-medication compared to drugs prescribed by physicians. The current narrative review was planned to focus on indicating prevalence rate of self-medication in different developed and under-developed countries, major risk factors and control of self-medication due to which antibiotic resistance rate can be minimised. The issue needs urgent attention of representative authorities for taking serious actions. Furthermore, arranging awareness seminars and implementing new policies/regulations to prevent the sale of any drug/antibiotic without prescription could play a vital role in bringing this alarming issue under control.

Antibiogram of ESBL and MBL producing Pseudomonas aeruginosa among the population of Hazara division, KPK, Pakistan.

OBJECTIVE: To investigate the frequency rate and sensitivity pattern of extended-spectrum beta-lactamase and metallobeta- lactamase producing Pseudomonas aeruginosa isolated from major hospitals. METHODS: The cross-sectional study was conducted in the Microbiology section of the Pathology Department of Ayub Medical College, Abbottabad, Pakistan, from September 2017 to April 2018, and comprised clinical samples collected from different medical wards of major hospitals in the study area. For the selective growth of Pseudomonas aeruginosa, Cetrimide agar was used, and different antibiotics were evaluated for the sensitivity pattern following Kirby-Bauer diffusion method. Pseudomonas aeruginosa producing extended-spectrum beta lactamase and metallo-beta-lactamase were identified through double disk synergy test and imipenem ethylenediaminetetraacetic acid tests respectively. Patient's demographic and medical history was noted on a proforma. Data was analysed using SPSS 22.0. RESULTS: Of the 242 samples screened, 46 (19%) were positive for Pseudomonas aeruginosa. These samples were highly sensitive to levofloxacin, amikacin, imipenem, meropenem and ciprofloxacin (p<0.05). Of the positive cases, 11 (23.91%) were detected for extended-spectrum beta-lactamase production, while 3 (6.52%) samples were detected for metallo-beta-lactamase production. CONCLUSIONS: Pseudomonas aeruginosa samples were widely resistant to most antibiotics, but were sensitive for some antibiotics which may be recommended by physicians when treating Pseudomonas aeruginosa infection.

Frequency and comparison among antibiotic resistant Staphylococcus aureus strains in selected hospitals of Peshawar, Pakistan.

OBJECTIVE: To assess and compare the frequency of antibiotic-resistant staphylococcus aureus strains. METHODS: A retrospective study was conducted at the privately-owned Welfare Medical Laboratory, Peshawar, Pakistan, and comprised record related to the period between 07th February 2017 and 23rd March 2018 of patients referred for pus-testing from Khyber Teaching Hospital, Lady Reading Hospital and the Hayatabad Medical Complex. Pus samples were obtained from various parts of body with cotton swabs. The samples were cultured and the isolated staphylococcus aureus strains were analysed against selected antibiotics. The frequency of the isolated strains was tested and compared using Prism 7 software. RESULTS: Of the 6780 samples, staphylococcus aureus was found in 4315(63.64%). Wild-type staphylococcus aureus strains were 2133(31.46%), followed by 825(12.16%) methicillin resistant, 792(11.68%) vancomycin intermediate, and 565(8.33%) vancomycin-resistant staphylococcus aureus strains. The isolated strains were significant (p<0.0001) for operated wounds, and non-significant (p=0.8915) for diabetic foot cases. CONCLUSIONS: The frequency of antibiotic-resistant staphylococcu saureus strains was high.

Identification of proteins responding to pathogen-infection in the red alga Pyropia yezoensis using iTRAQ quantitative proteomics.

BACKGROUND: Pyropia yezoensis is an important marine crop which, due to its high protein content, is widely used as a seafood in China. Unfortunately, red rot disease, caused by Pythium porphyrae, seriously damages P. yezoensis farms every year in China, Japan, and Korea. Proteomic methods are often used to study the interactions between hosts and pathogens. Therefore, an iTRAQ-based proteomic analysis was used to identify pathogen-responsive proteins following the artificial infection of P. yezoensis with P. porphyrae spores. RESULTS: A total of 762 differentially expressed proteins were identified, of which 378 were up-regulated and 384 were down-regulated following infection. A large amount of these proteins were involved in disease stress, carbohydrate metabolism, cell signaling, chaperone activity, photosynthesis, and energy metabolism, as annotated in the KEGG database. Overall, the data showed that P. yezoensis resists infection by inhibiting photosynthesis, and energy and carbohydrate metabolism pathways, as supported by changes in the expression levels of related proteins. The expression data are available via ProteomeXchange with the identifier PXD009363. CONCLUSIONS: The current data provide an overall summary of the red algae responses to pathogen infection. This study improves our understanding of infection resistance in P. yezoensis, and may help in increasing the breeding of P. porphyrae-infection tolerant macroalgae.

Effects of sodium bicarbonate concentration on growth, photosynthesis, and carbonic anhydrase activity of macroalgae Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae (Gracilariales, Rhodophyta).

There is potential for bicarbonate to improve crop yields and economic efficiency of marine algae. However, few studies have focused on the effect of bicarbonate on the growth, photosynthesis, and enzyme activity associated with carbon utilization, especially in commercial macroalgae. Here, the addition of bicarbonate (up to 420 mg L(-1)) to macroalgal cultures has been evaluated for Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae with respect to growth rate, photosynthetic activity, carbonic anhydrase activity, and biochemical composition. The results showed that the effects of NaHCO3 on growth, chlorophyll a, phycoerythrin, photosynthetic oxygen evolution, photochemical parameters of PSI and PSII, carbonic anhydrase activity, and nitrogen content were significant (P < 0.05) and followed the same pattern in the three species. The parameter values were promoted in lower NaHCO3 concentrations (up to 252 or 336 mg L(-1)) and inhibited in higher NaHCO3 concentrations (>336 mg L(-1) for Gp. lemaneiformis and >420 mg L(-1) for the other two species). Moreover, species-specific differences induced by supplementation with bicarbonate were discovered during culture. Optimal concentrations of NaHCO3 used in this study were 252 mg L(-1) for Gp. lemaneiformis and 336 mg L(-1) for G. vermiculophylla and G. chouae. These results suggest that an adequate supplementation of sodium bicarbonate is a viable strategy for promoting growth and photosynthetic activity in some macroalgae as well as for improving biochemical composition. The study will help to accelerate the growth rate of algae and improve the quality of thalli, and will also be useful for enhancing the understanding of carbon utilization in macroalgae.

Lipase and Protease Production Ability of Multi-drug Resistant Bacteria Worsens the Outcomes of Wound Infections.

BACKGROUND: Surgical site infections are one of the major clinical problems in surgical departments that cost hundreds of millions of dollars to healthcare systems around the world. AIM: The study aimed to address the pressing issue of surgical site infections, which pose significant clinical and financial burdens on healthcare systems globally. Recognizing the substantial costs incurred due to these infections, the research has focused on understanding the role of lipase and protease production by multi-drug resistant bacteria isolated from surgical wounds in the development of post-surgical wound infections. METHODS: For these purposes, 153 pus specimens were collected from patients with severe post-surgical wound infections having prolonged hospital stays. The specimens were inoculated on appropriate culture media. Gram staining and biochemical tests were used for the identification of bacterial growth on suitable culture media after 24 hours of incubation. The isolated pathogens were then applied for lipase and protease, key enzymes that could contribute to wound development, on tributyrin and skimmed milk agar, respectively. Following the CSLI guidelines, the Kirby-Bauer disc diffusion method was used to assess antibiotic susceptibility patterns. The results revealed that a significant proportion of the samples (127 out of 153) showed bacterial growth of Gram-negative (n = 66) and Gram-positive (n = 61) bacteria. In total, isolated 37 subjects were declared MDR due to their resistance to three or more than three antimicrobial agents. The most prevalent bacteria were Staphylococcus aureus (29.13%), followed by S. epidermidis (18.89%), Klebsiella pneumoniae (18.89%), Escherichia coli (14.96%), Pseudomonas aeruginosa (10.23%), and Proteus mirabilis (7.87%). Moreover, a considerable number of these bacteria exhibited lipase and protease activity with 70 bacterial strains as lipase positive on tributyrin agar, whereas 74 bacteria showed protease activity on skimmed milk agar with P. aeruginosa as the highest lipase (69.23%) and protease (76.92%) producer, followed by S. aureus (lipase 62.16% and protease 70.27%). RESULTS: The antimicrobial resistance was evaluated among enzyme producers and non-producers and it was found that the lipase and protease-producing bacteria revealed higher resistance to selected antibiotics than non-producers. Notably, fosfomycin and carbapenem were identified as effective antibiotics against the isolated bacterial strains. However, gram-positive bacteria displayed high resistance to lincomycin and clindamycin, while gram-negative bacteria were more resistant to cefuroxime and gentamicin. CONCLUSION: In conclusion, the findings suggest that lipases and proteases produced by bacteria could contribute to drug resistance and act as virulence factors in the development of surgical site infections. Understanding the role of these enzymes may inform strategies for preventing and managing post-surgical wound infections more effectively.

True prevalence of twin HDV-HBV infection in Pakistan: a molecular approach.

Hepatitis Delta Virus (HDV) infects only patients that are already infected by hepatitis B virus (HBV) because this is sub satellite virus which depends on and propagate only in the presence of HBV. HDV causes co-infection or super infection with sever complication as compared to only HBV infection. No study on molecular level on HDV is available from this region; therefore, the aim of this study was to found out the molecular epidemiology of HDV (as a co-infection with HBV) in different geographical regions of Pakistan.Total 228 HBsAg positive samples were received for the study from different geographical regions of the country. Only HBV DNA PCR positive samples were further utilized for the presence of HDV RNA. For this purpose, HDV RNA and HBV DNA was extracted and amplified using reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR and real-time PCR. Out of the total 228 HBsAg positive samples, HBV DNA was detected in total 190 (83.3%) samples belonged to different patients. Of these 190 patients, HDV RNA was observed in 53 (28%) patients. Of the 53 HDV positive cases, 37 (69.8%) were males and 16 (30.2%) were female patients. The percentage of dual infection was found higher significantly (p < 0.05) in male patients as compared to female patients. Total 41 (26.8%) patients were below 40 years and 13 (31.7%) were above 40 years of age. No significant difference was seen in patients with ages above or below 40 years. In the provinces of Sindh, Khyber Pakhtoonkhaw and Punjab the observed prevalence of HDV was 67%, 6% and 4% respectively.In conclusion, the HDV infection is not uncommon in Pakistan and its prevalence is higher significantly in the Province of Sindh (p < 0.01) and male six (p < 0.05).

Preparation, characterization and stability studies of cross-linked α-amylase aggregates (CLAAs) for continuous liquefaction of starch.

In current study, α-amylase of fungal origin was immobilized using cross-linking strategy. The influence of precipitant (ammonium sulphate) and cross-linker (glutaraldehyde) concentration revealed that 60% (w/v) precipitant and 1.5% (v/v) cross-linker saturation was required to attain optimum activity. Cross-linked amylase aggregates (CLAAs) were characterized and 10-degree shift in optimum temperature (soluble enzyme: 50 °C; cross-linked: 60 °C) and 1-unit shift in pH (soluble enzyme: pH -6; cross-linked: pH -7) was observed after immobilization. The Vmax for soluble α-amylase and its cross-linked form was 1225 U ml-1 and 3629 U ml-1, respectively. The CLAAs was more thermostable than its soluble form and retained its 30% activity even after 60 min of incubation at 70 °C. Moreover, cross-linked amylase retained its activity after two months while its soluble counterpart lost its complete activity after 10 and 20 days at 30 °C and 4 °C storage, respectively. Reusability test showed that cross-linked amylase could retain 13% of its residual activity after 10 repeated cycles. Therefore, 10 times more glucose was produced after cross-linking than soluble amylase when it was utilized multiple times. This study indicates that amylase aggregates are highly effective for continuous liquefaction of starch, hence have strong potential to be used for different industrial processes.

Biological Transformation of Zearalenone by Some Bacterial Isolates Associated with Ruminant and Food Samples.

Zearalenone (ZEA) is a secondary metabolite produced by Fusarium spp., the filamentous fungi. Food and feed contamination with zearalenone has adverse effects on health and economy. ZEA degradation through microorganisms is providing a promising preventive measure. The current study includes isolation of 47 bacterial strains from 100 different food and rumen samples. Seventeen isolates showed maximum activity of ZEA reduction. A bacterial isolate, RS-5, reduced ZEA concentration up to 78.3% through ELISA analysis and 74.3% as determined through HPLC. Ten of the most efficient strains were further selected for comparison of their biodegradation activity in different conditions such as incubation period, and different growth media. The samples were analyzed after 24 h, 48 h, and 72 h of incubation. De Man Rogosa Sharp (MRS) broth, Tryptic soy broth, and nutrient broth were used as different carbon sources for comparison of activity through ELISA. The mean degradation % ± SD through ELISA and HPLC were 70.77% ± 3.935 and 69.11% ± 2.768, respectively. Optimum reducing activity was detected at 72 h of incubation, and MRS broth is a suitable medium. Phylogenetic analysis based on 16S rRNA gene nucleotide sequences confirmed that one of the bacterial isolate RS-5 bacterial isolates with higher mycotoxin degradation is identified as Bacillus subtilis isolated from rumen sample. B05 (FSL-8) bacterial isolate of yogurt belongs to the genus Lactobacillus with 99.66% similarity with Lactobacillus delbrukii. Similarly, three other bacterial isolates, D05, H05 and F04 (FS-17, FSL-2 and FS-20), were found to be the sub-species/strains Pseudomonas gessardii of genus Pseudomonas based on their similarity level of (99.2%, 96% and 96.88%) and positioning in the phylogenetic tree. Promising detoxification results were revealed through GC-MS analysis of RS-5 and FSL-8 activity.

Detection, characterization and expression dynamics of histone proteins in the dinoflagellate Alexandrium pacificum during growth regulation.

Histones are the most abundant proteins associated with eukaryotic nuclear DNA. The exception is dinoflagellates, which have histone protein expression that is mostly reported to be below detectable levels. In this study, we investigated the presence of histone proteins and their functions in the dinoflagellate, Alexandrium pacificum. Histone protein sequences were analyzed, focusing on phylogenetic analysis and histone code. Histone expression was analyzed during the cell cycle and under nutritionally enhanced conditions using quantitative-PCR and western blots. Acid-soluble proteins were subjected to mass spectrometry analysis. To our knowledge, this is the first report of immunological detection of histone proteins (H2B and H4) in any dinoflagellate species. Absolute quantification of histone transcript in activily dividing cells revealed significant transcription in cells. The stable expression of histones during the cell cycle suggested that the histone genes in A. pacificum belonged to a replication-independent class and appeared to have a limited role in DNA packaging. The conservation of numerous post-translationally modified residues of multiple histone variants and differential expression of histones under nutritionally enhanced conditions suggested their functional significance in dinoflagellates. However, we detected histone H2B protein only via mass spectrometry. Histone-like protein was identified as most abundant acid-soluble protein of the cells.

Distinctive Nuclear Features of Dinoflagellates with A Particular Focus on Histone and Histone-Replacement Proteins.

Dinoflagellates are important eukaryotic microorganisms that play critical roles as producers and grazers, and cause harmful algal blooms. The unusual nuclei of dinoflagellates "dinokaryon" have led researchers to investigate their enigmatic nuclear features. Their nuclei are unusual in terms of their permanently condensed nucleosome-less chromatin, immense genome, low protein to DNA ratio, guanine-cytosine rich methylated DNA, and unique mitosis process. Furthermore, dinoflagellates are the only known group of eukaryotes that apparently lack histone proteins. Over the course of evolution, dinoflagellates have recruited other proteins, e.g., histone-like proteins (HLPs), from bacteria and dinoflagellates/viral nucleoproteins (DVNPs) from viruses as histone substitutes. Expression diversity of these nucleoproteins has greatly influenced the chromatin structure and gene expression regulation in dinoflagellates. Histone replacement proteins (HLPs and DVNPs) are hypothesized to perform a few similar roles as histone proteins do in other eukaryotes, i.e., gene expression regulation and repairing DNA. However, their role in bulk packaging of DNA is not significant as low amounts of proteins are associated with the gigantic genome. This review intends to summarize the discoveries encompassing unique nuclear features of dinoflagellates, particularly focusing on histone and histone replacement proteins. In addition, a comprehensive view of the evolution of dinoflagellate nuclei is presented.

Mitochondrial genome of Opsaridium microlepis (cypriniformes: cyprinidae).

The complete mitochondrial genome of Opsaridium microlepis was determined by using llumina sequencing method. The genome is 168 25 bp in length, comprising 13 protein-coding genes, 20 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and one control region (D-loop region). Most genes are encoded on the heavy strand (H-strand), except for nad6 and eight tRNA genes. Base composition of the H-strand are A (27.97%), C (26.34%), G (18.37%) and T (27.32%) with AT bias of 55.29%.

Molecular cloning and sequence analysis of a PVGOX gene encoding glucose oxidase in Penicillium viticola F1 strain and it's expression quantitation.

The PVGOX gene (accession number: KT452630) was isolated from genomic DNA of the marine fungi Penicillium viticola F1 by Genome Walking and their expression analysis was done by Fluorescent RT-PCR. An open reading frame of 1806bp encoding a 601 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 65,535.4 was characterized. The deduced protein showed 75%, 71%, 69% and 64% identity to those deduced from the glucose oxidase (GOX) genes from different fungal strains including; Talaromyces variabilis, Beauveria bassiana, Aspergillus terreus, and Aspergillus niger, respectively. The promoter of the gene (intronless) had two TATA boxes around the base pair number -88 and -94 and as well as a CAAT box at -100. However, the terminator of the PVGOX gene does not contain any polyadenylation site (AATAAA). The protein deduced from the PVGOX gene had a signal peptide containing 17 amino acids, three cysteine residues and six potential N-linked glycosylation sites, among them, -N-K-T-Y- at 41 amino acid, -N-R-S-L- at 113 amino acid, -N-G-T-I- at 192 amino acid, -N-T-T-A at 215 amino acid, -N-F-T-E at 373 amino acid and -N-V-T-A- at 408 amino acid were the most possible N-glycosylation sites. Furthermore, the relative transcription level of the PVGOX gene was also stimulated in the presence of 4% (w/v) of calcium carbonate and 0.5 % (v/v) of CSL in the production medium compared with that of the PVGOX gene when the fungal strain F1 was grown in the absence of calcium carbonate and CSL in the production medium, suggesting that under the optimal conditions, the expression of the PVGOX gene responsible for gluconic acid biosynthesis was enhanced, leading to increased gluconic acid production. Therefore, the highly glycosylated oxidase enzyme produced by P. viticola F1 strain might be a good producer in the fermentation process for the industrial level production of gluconic acid.

Determination of hepatitis C virus genotypes circulating in different districts of Punjab (Pakistan).

INTRODUCTION: Hepatitis C virus (HCV) is a principal cause of severe liver diseases worldwide and a possible source of significant morbidity and fatality in the long term. Information on the genotype is more significant because it has prognostic value in the response to antiviral therapy. OBJECTIVES: This study aimed to determine the frequency of various HCV genotypes circulating in the different districts of Punjab and to show the pattern distribution of HCV genotypes in different age groups and sexes. MATERIALS AND METHODS: A total of 542 HCV-positive patients were selected from various districts of the province and were subjected to an HCV genotype-specific assay. Among 542 positive patients, 300 (55.35%) were men and 242 (44.65%) were women. There were 245 (45.20%), 61 (11.25%), 56 (10.33%), 56 (10.33%), 53 (9.77%), 27 (5%), 20 (3.69%), 16 (3%), and eight (1.47%) HCV-positive patients from Lahore, Sargodha, Multan, Toba Tek Singh, Faisalabad, Rawalpindi, Mandi Bahauddin Gujranwala, and Sahiwal districts, respectively. RESULTS: Of a total of 542 serum samples analyzed, 476 (88%) were successfully genotyped whereas 66 (12%) samples were undetermined genotypes. Among the typable genotypes, 1a was found in 37 (7%), 1b in 18 (3%), 3a in 386 (71%), and genotype 3b in five (1%) patients. Thirty patients (6%) were identified to be infected with mixed HCV genotypes. Genotypes 3a (P=0.0001), 1a (P=0.001), and untypable genotypes (P=0.03) were circulating significantly in all the studied districts. All the genotypes were distributed evenly in male and female patients. The most affected age range of patients was 21-40 years as compared with teenage and older age groups. CONCLUSION: The study found a significantly high prevalence of HCV among the patients of Punjab. In addition, genotype 1a was found to be a significantly and rapidly increasing genotype in the study area. It appears that HCV-3a (the most prevalent genotype) may be replaced by genotype 1a. If this occurs, it will make the present scenario more complex in terms of response to therapy and economic burden on the health sector of Pakistan. HCV infection is more common in the age group of 21-40 years. All the genotypes were distributed at the same frequency in men and women.