Dual application of black soldier fly (Hermetia illucens): Protein-rich animal feed and biological extraction agent for polyhydroxybutyrate

Aims@#The primary aim of the present study is to evaluate the effect of rearing substrates on the nutritional content of black soldier fly larvae (BSFL) by incorporating Cupriavidus necator cells containing intracellular polyhydroxybutyrate (PHB) in BSFL diet to further increase the protein content and simultaneously to biologically extract the polymer by utilizing the digestive system of BSFL. The potential application of BSFL as a biological PHB extraction agent was determined.@*Methodology and results@#Two feeding strategies consists of a mixture of protein (P) to carbohydrate (C) with a ratio of P50:C50 food waste (control feeding) and feed with bacterial cells (modified feeding). A comparison on the proximate analysis between this research and two commercially available products were conducted. Feeding BSFL with P50:C50 food waste revealed the highest crude protein content of 81.3 ± 0.2%. Additional bacteria cells in the BSFL diet, however, showed a negligible decrease in crude protein content of 0.67% as compared to the control feeding. Howbeit, this results comparably higher in contrast to the commercial products, with increment of crude protein content by 12.1% and 40.8%, respectively.@*Conclusion, significance and impact of study@#Two desirable products were obtained from the feeding with cells: (1) high protein content of BSFL and (2) biologically extracted polymer. This is the first study to demonstrate the utilization of BSFL as a biological extraction agent to partially extract biopolymer and increase the protein content by feeding with cells.

Maternal and Fetal Outcome of Cardiac Disease in Pregnancy: A Retrospective Study at Tertiary Institute

Introduction: Cardiac disease in pregnancy is still a major cause of maternal and fetal mortality. Although the reported incidence varies between 0.1 and 4%, 1–3 cardiac disease remains a significant cause of maternal death worldwide. The incidence of cardiac disease during pregnancy has remained stable for many years even with a significant decrease in the occurrence of rheumatic heart disease (RHD) as this decrease is being compensated by significant increase of pregnancy in women with congenital heart disease (CHD). Therefore, in this study, we aim to analyze the incidence of cardiac disease in pregnancy and to assess the maternal and fetal outcome. Materials and Methods: The retrospective study was carried out in 47 women with cardiac disease in a tertiary institute over a period of 2 years. Results: In the present study, incidence of cardiac disease at our centre was 0.081%. RHD was the most common heart disease in pregnancy (70.21%) followed by CHD (23.40%) and peripartum cardiomyopathy (6.38%). Among RHD, mitral valve stenosis was most common followed by mitral stenosis with mitral regurgitation. Number of vaginal deliveries was 36 and cesarean was done in 11 patients. Conclusions: A cardiac disease is a high-risk pregnancy. It is a multidisciplinary teamwork to have optimal maternal and fetal outcome. Hence, constant vigilance is required throughout antenatal, intrapartum, and postpartum period to avoid adverse outcomes.

Efficient biosynthesis and recovery of polyhydroxyalkanoate

Petrochemical-derived plastics have become a source of pollution for decades, and finding alternative plastics that are environmentally friendly has become a matter of urgency. Polyhydroxyalkanoate (PHA), a biopolyester synthesized by microbial cells, has properties that make it suitable as a biodegradable plastic material. The diversity of PHA makes it applicable to a wide range of products, from packaging to biomedical devices. The main challenge in commercialization of PHA is the cost of production. Although many studies have been focused on obtaining high yields of PHA, up until now, there is no absolute definition of efficient production of PHA, as there are many factors that could contribute to the efficiency of a process. Efficiency in PHA recovery also contributes to the commercial viability of PHA production. This review focuses on the efficiency of PHA biosynthesis from several aspects relating to the criteria for efficient production. The development of new strategies for improved production, including utilization of low cost carbon sources, genetic modification of PHA-producing microbes, and fermentation strategies are discussed here. Advances in recovery of PHA, as well as the potential of biological recovery techniques, are also highlighted in this review.

Screening of bacteria for PHA production using waste glycerol as carbon source and the ability of new strain to produce P(3HB-co-3HV) copolymer

Aims: This study is focused on the isolation, characterization and screening of new bacterial strains isolated from soil and wastewater samples that are able to produce PHA utilizing waste glycerol as sole carbon source in order to create useful products from waste glycerol and at the same time reduce the PHA production cost. A new isolate, Burkholderia contaminans Kad1 strain was investigated for its ability to biosynthesize PHA copolymers containing 3HV monomers from waste glycerol and 3-hydroxyvalerate (3HV) precursors. Methodology and results: PHA producing bacteria were screened using Nile Red and 1% of Nile Blue method. The presence of PHA granules was detected using phase contrast and fluorescence microscopy. Burkholderia contaminans Kad1, one out of 23 positive samples, was selected for further study because of its ability to produce high PHA content (47 wt%) and dry cell weight (DCW), (4.2 g/L) when waste glycerol 2% (v/v) was used as the sole carbon source. The 16S rDNA and the PHA synthase gene were sequenced and the PHA produced was confirmed by NMR analysis. A mixture of waste glycerol and sodium valerate fed to the culture gave rise to poly(3-hydroxybutyrate-co-3- hydroxyvalerate) [P(3HB-co-3HV)]. The mole fraction of 3HV monomer in the co-polymer P(3HB-co-3HV) sample analyzed using 1H NMR was 23 mol%. Conclusion, significance and impact of study: This study demonstrated for the first time B. contaminans Kad1 was able to use waste glycerol for PHA biosynthesis including the P(3HB-co-3HV) copolymer using a mixture of waste glycerol with sodium valerate as the precursor.

Isolation and identification of bacteria and fungi growing spontaneously on polyhydroxyalkanoate pellets recovered by a new biological process

Aims: The study was carried out to isolate and identify the spontaneously growing populations of bacteria and fungi on the surface of biologically recovered polyhydroxyalkanoate (PHA) copolymer, poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) [P(3HB-co-3HHx)]. Methodology and results: Large-scale PHA biosynthesis was carried out using 300 L fermenter and a biological methodology developed in our laboratory was utilized for PHA recovery. Using standard microbiological and molecular biology techniques the naturally growing microbial populations on the surface of biologically recovered PHA were identified. Scanning electron microscopy (SEM) analysis showed that the identified bacterial (Bacillus cereus and Burkholderia cepacia) and fungal isolates (Aspergillus niger, Byssochlamys nivea, Penicillium citrinum and Penicillium griseofulvum) were able to grow on and degrade the P(3HB-co-3HHx) copolymer. Conclusion, significance and impact of study: This is the first report on biologically recovered PHA pellet addressing the occurrence of microorganisms that grew spontaneously on it during storage under laboratory conditions. Fungi appeared to be dominant over bacteria in their ability to colonize the biologically recovered PHA.

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

Characteristics of limestone soil collected from Gunung Lang, Perak and metagenomic analysis of the soil microbial community

Aims: This project was aimed to study the microbial diversity of the limestone soil and its correlation with the environment. Methodology and results: The study was carried out using samples obtained from Gunung Lang, Ipoh, Perak in August 2013. X-ray diffraction analysis of the rock structure confirmed that the samples were of limestone origin. Besides that, soil analysis revealed that this area was fertile and rich in nutrients. It therefore served as a suitable habitat for microorganismal diversity to flourish. This was proven by the 16S rDNA metagenomic analysis which targeted on 16S rDNA variable region V3-V5 using Illumina MiSeq sequencer. Using this approach, a variety of microorganisms was identified and many yet to be characterized microorganisms were detected from this area. Conclusion, significance and impact of study: This is the first study in Malaysia that aimed to study the microbial diversity of limestone soils through metagenomic approach. The study showed that limestone is rich in microbial diversity and it is a place worth looking for novel microbes and genes of interest in biotechnology.

Biosynthesis of P(3HB-co-3HV-co-3HHp) terpolymer by Cupriavidus necator PHB4 transformant harboring the highly active PHA synthase gene of Chromobacterium sp. USM2

Aims: This study evaluates potentials of Cupriavidus necator PHB4 transformant harboring the highly active polyhydroxyalkanoate synthase gene (phaC) of a locally isolated Chromobacterium sp. USM2 for its ability to incorporate 3-hydroxyheptanoate (3HHp) monomer. Methodology and results: A mixture of fructose and sodium heptanoate fed to the culture gave rise to poly(3- hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyheptanoate), [P(3HB-co-3HV-co-3HHp)] terpolymer synthesis, with traces of 3HHp monomers confirmed through gas chromatography (GC), proton (1H) and carbon (13C) NMR spectra. Conclusion, significance and impact of study: This study has revealed that the PHA synthase of Chromobacterium sp. USM2 has a broad range of substrate specificity. The synthase is able to polymerize 3-hydroxyalkanoate monomers having 4–7 carbon atoms.

Waste cooking oil as substrate for biosynthesis of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate): Turning waste into a value-added product

Aims: Improper disposal of domestic wastes, such as waste cooking oil (WCO), contributes to the deterioration of the environment and may lead to health problems. In this study, we evaluated the potential of plant-based WCO as a carbon source for the commercial biosynthesis of the bio-plastics, poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). The consumption of WCO for this purpose would mitigate their pollution of the environment at the same time. Methodology and Results: WCO collected from several cafeterias in USM was tested as the carbon source for polyhydroxyalkanoates (PHA) production. A selection of suitable nitrogen source was first conducted in order to obtain an acceptable number of dry cell weight (DCW) and PHA content. Urea was found to be a suitable nitrogen source for the two bacterial strains used in our study, Cupriavidus necator H16 and its transformed mutant, C. necator PHB¯4 harboring the PHA synthase gene of Aeromonas caviae (PHB¯4/pBBREE32d13). With WCO as the sole carbon source, C. necator H16 yielded a relatively good dry cell weight (DCW=25.4 g/L), with 71 wt% poly(3-hydroxybutyrate) P(3HB) content. In comparison, the DCW obtained with fresh cooking oil (FCO) was 24.8 g/L. The production of poly(3 hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from WCO by the transformant C. necator PHB¯4 was comparable, yielding a DCW of 22.3 g/L and P(3HB-co-3HHx) content of 85 wt%. Lipase activities for both bacterial strains reached a maximum after 72 h of cultivation when time profile was conducted. Conclusion, significance and impact of study: The use of WCO as a carbon source in the biosynthesis of the bioplastic, PHA, turns a polluting domestic waste into a value-added biodegradable product. This renewable source material can thus be exploited for the low cost production of PHA.

In vitro expression studies of non structural 1 protein of Canine Parvo virus 2 by polyclonal antiserum raised against CPV2-NS1 protein expressed in Escherichia coli as an antigen.

The canine Parvovirus 2, non-structural 1(NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1(rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS–PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 × His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.

Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies.

In the present study recombinant VP3 (rVP3) was expressed in E.coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS–PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37ºC. The 6×His-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.

Waardenburg syndrome: A rare genetic disorder, a report of two cases.

Waardenburg syndrome (WS) is a rare genetic disorder. Patients have heterochromia or eyes with iris of different color, increased inter-canthal distance, distopia canthorum, pigmentation anomalies, and varying degree of deafness. It usually follows autosomal dominant pattern. In this report, two cases have been discussed but no familial history of WS has been found. Counseling of the patient is necessary and cases of irreversible deafness have been treated.

Biosynthesis and Lipase-Catalysed Hydrolysis of 4-Hydroxybutyrate-Containing Polyhydroxyalkanoates from Delftia acidovorans

Aims: Polyhydroxyalkanoates (PHA) having various molar fractions of 4-hydroxybutyrate has been successfully synthesized by Delftia acidovorans. Methodology and results: The monomer compositions of the PHA were varied by cultivating the bacterium in a mixture of 1,4-butanediol and sodium valerate, γ-butyrolactone and sodium valerate as well as 4-hydroxybutyric acid and sodium valerate, which resulted in the production of PHA terpolymers. Although the highest terpolymer content achieved was only 57 wt% of the dry cell weight, the 4HB molar fractions can be regulated from 2-50 mol% when culture conditions such as initial pH, inoculum concentration and aeration were varied. The in vitro degradation of [P(3HB-co-50 % 4HB)]synthesized by D. acidovorans were also studied by monitoring the erosion rate of the copolymer in aqueous solutions of lipases (Lipase A ‘Amano’ 12 and Newlase F). Results have shown that the types of lipases, concentration of lipase solution and pH of the buffer solution influenced the degradation rate of the PHA copolymer. Conclusion, significance and impact of the study: The overall results have shown that D. acidovorans is a very promising strain for the production of 4HB containing PHAs with specific compositions which are very suitable to be tailor made into biodegradable and biocompatible materials for medical applications.

Characterization and in vitro expression of non-structural 1 protein of canine parvovirus (CPV-2) in mammalian cell line.

Parvoviruses are small, 260-Å-diameter, icosahedral, non-enveloped, single-stranded DNA viruses with a genome of approximately 5 kb. Non structural protein, (NS-1) is especially relevant, being both essential for virus replication and the main factor responsible for virus pathogenicity and cytotoxicity. This protein has also been reported to possess the property of killing of transformed cells. The present study was carried out to clone, characterize and express the NS-1 gene of canine parvovirus. NS-1 complete CDS 2020bp was amplified, cloned into eukaryotic expression vector pcDNA 3.1(+), sequenced and characterized by in vitro expression analysis. Functional activity of recombinant construct, pcDNA.cpv.NS-1, was evaluated by RT-PCR and flow cytometry for the expression of NS-1 specific mRNA and NS-1 protein, respectively, in transfected HeLa cells. This recombinant plasmid may serve as an important tool to evaluate the apoptotic potential of NS-1 protein of canine parvovirus in cultured HeLa cells.

Development and in vitro characterization of a bivalent DNA containing HN and F genes of velogenic Newcastle disease virus.

Newcastle disease (ND) is highly contagious, economically important viral disease affecting most of avian species worldwide. Newcastle disease virus (NDV) has single stranded negative sense RNA genome which encodes for six structural and two non-structural proteins. Envelope glycoproteins i.e. hemagglutinin-neuraminidase (HN) and the fusion (F), elicit protective immune response. In this study, HN and F genes of velogenic (virulent) strain were amplified and cloned at multiple cloning sites A and B, respectively into pIRES bicistronic vector for use as bivalent DNA vaccine against ND. The recombinant plasmid was characterized for its orientation by restriction enzyme digestion and PCR. Expression of HN and F genes was assessed in transfected Vero cells at RNA level using RT-PCR in total RNA as well as protein level using IFAT, IPT and western blot using NDV specific antiserum. All these experiments confirmed that HN and F genes cloned in recombinant pIRES.nd.hn.f are functionally active. The recombinant construct is being evaluated as DNA vaccine against ND.

HIV/AIDS in Families.

HIV/AIDS is a disease that affects families in a profound and tragic way affecting family structure by erasing decades of health, economic, social progress and reducing life expectancy. There is limited empirical data on HIV/AIDS affected families.The present one and half year prospective study was conducted to identify HIV/AIDS existing in families. HIV/AIDS cases were diagnosed as per the NACO, 2000 criteria.The present study enrolled a total 230 HIV/AIDS patients. 65.21 % (150) of the patients were married and 34.78% (80) were unmarried. Among total unmarried population 27.39% (63) and 6.52% (17) were adults and pediatric population respectively. Among total pediatric population 4 were orphan, 3 were partially orphan with mother alive and 10 were with both parents alive but both positive for HIV/AIDS. Among total married 13.04% (30) were widow/widowers who had lost their spouse, whereas those living with live partner were 52.17% (120) forming a total of 60 pairs of couples. Among these 60 couples 40 of them were both positive for HIV/AIDS, in 15, single partner was positive with interesting finding in one case where male was negative and female was positive and mode of transmission was unclear and in another 5 status of spouse was not known due to unknown reasons. Among 40 couples where both partners were affected, 3 couples were isolated where complete family ie all children were affected by HIV/AIDS; in another 20 couples children’s were not affected; in 4 couples children were partially affected i.e. some children’s were affected; in 9 couples status of their children’s were not evident and another 4 couples were without any issue.The results of present study suggest HIV/ AIDS affects whole family and not an individual and thus whole family should be screened, evaluated, treated and educated for HIV/ AIDS.

Megakaryocytic Aplasia.

Acquired Megakaryocytic aplasia is a rare disorder defined by severe thrambocytopenia with no other haematological and absent or severely marow megakaryocytes.