Targeting Microbial Biofouling by Controlling Biofilm Formation and Dispersal Using Rhamnolipids on RO Membrane.

Finding new biological ways to control biofouling of the membrane in reverse osmosis (RO) is an important substitute for synthetic chemicals in the water industry. Here, the study was focused on the antimicrobial, biofilm formation, and biofilm dispersal potential of rhamnolipids (RLs) (biosurfactants). The MTT assay was also carried out to evaluate the effect of RLs on biofilm viability. Biofilm was qualitatively and quantitatively assessed by crystal violet assay, light microscopy, fluorescence microscopy (bacterial biomass (µm2), surface coverage (%)), and extracellular polymeric substances (EPSs). It was exhibited that RLs can reduce bacterial growth. The higher concentrations (≥100 mg/L) markedly reduced bacterial growth and biofilm formation, while RLs exhibited substantial dispersal effects (89.10% reduction) on preformed biofilms. Further, RLs exhibited 79.24% biomass reduction while polysaccharide was reduced to 60.55 µg/mL (p < 0.05) and protein to 4.67 µg/mL (p < 0.05). Light microscopy revealed biofilm reduction, which was confirmed using fluorescence microscopy. Microscopic images were processed with BioImageL software. It was revealed that biomass surface coverage was reduced to 1.1% at 1000 mg/L of RLs and that 43,245 µm2 of biomass was present for control, while biomass was reduced to 493 µm2 at 1000 mg/L of RLs. Thus, these data suggest that RLs have antimicrobial, biofilm control, and dispersal potential against membrane biofouling.

The role of pricing strategies, clean technologies, and ecological regulation on the objectives of the UN 2030 Agenda.

The dynamics of global emissions and the increasing focus on market-based policy instruments have prompted this research to examine the extent to which such instruments are successful in emission control. The study explores the influence of carbon taxes, eco-friendly innovations, and ecological policy in attaining sustainable development goals and fulfilling the mitigation targets of climate change for 2030. The article selected 15 EU countries from southern and western regions and tested the empirical relationship between 2000 and 2018. This work used second-generation testing approaches and error correction-based modeling approaches to analyze the relationship between the variables. The results show that eco-friendly innovations and environmental policies help reduce emissions in the long and short run. On the other hand, carbon taxes have a more prominent effect on mitigation efforts, specifically in the short run. Factors such as urbanization, economic growth, and energy consumption are the most prominent polluting elements, the results being consistent in all models. The results further show a unidirectional and bidirectional causality relationship between the variables, and outcomes are more country-specific. Given these arguments, carbon taxes are a short-term instrument in combating carbon emissions. However, the sustainable development vision 2030 relies on eco-innovations linked with research and development and the transition from gray to green energy.

The role of green innovations, environmental policies and carbon taxes in achieving the sustainable development goals of carbon neutrality.

The green innovations, environmental policies, and carbon taxes are the tools to achieve sustainable development goals (SDGs) in the mitigation process. This study is intended to examine the impact of innovation, carbon pricing (CTAX), environmental policies (EP), and energy consumption (ECON) on PM2.5 and greenhouse gas (GHG) emission for Central-Eastern European countries. The panel effect during 2000-2018 is tested using a dynamic panel data model while the Granger causality approach obtains country-related outcomes. The outcomes reveal that eco-friendly innovations have a more profound effect on carbon mitigation. Environmental policies reduce emissions by 2.7% in the short run and 17.4% in the long run. Similarly, CTAX mitigates GHG emissions by 8.6% in the short-run and PM2.5 by 0.9% and 5.7% in the short and long run. However, urbanization, energy consumption and trade openness are the leading polluters in the region. The main findings remain dominant in the country-specific results and find unidirectional and bidirectional causality evidence among variables. The research concludes that green innovations and strict environmental policy can lead towards achieving sustainable development goals using carbon taxes as a tool on the way.

Combating Biofilm by Targeting Its Formation and Dispersal Using Gallic Acid against Single and Multispecies Bacteria Causing Dental Plaque.

Exploring biological agents to control biofilm is a vital alternative in combating pathogenic bacteria that cause dental plaque. This study was focused on antimicrobial, biofilm formation and biofilm dispersal efficacy of Gallic acid (GA) against bacteria, including Proteus spp., Escherichia coli, Pseudomonas spp., Salmonella spp., Streptococcus mutans, and Staphylococcus aureus and multispecies bacteria. Biofilm was qualitatively and quantitatively assessed by crystal violet assay, florescence microscopy (bacterial biomass (µm2), surface coverage (%)) and extracellular polymeric substances (EPS). It was exhibited that GA (1-200 mg/L) can reduce bacterial growth. However, higher concentrations (100-200 mg/L) markedly reduced (86%) bacterial growth and biofilm formation (85.5%), while GA did not exhibit any substantial dispersal effects on pre-formed biofilm. Further, GA (20-200 mg/L) exhibited 93.43% biomass reduction and 88.6% (p < 0.05) EPS (polysaccharide) reduction. Microscopic images were processed with BioImageL software. It was revealed that biomass surface coverage was reduced to 2% at 200 mg/L of GA and that 13,612 (µm2) biomass was present for control, while it was reduced to 894 (µm2) at 200 mg/L of GA. Thus, this data suggest that GA have antimicrobial and biofilm control potential against single and multispecies bacteria causing dental plaque.

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.

Stunting and associated factors in children of less than five years: A hospital-based study.

OBJECTIVES: To estimate frequency of stunting and associated factors in children aged less than five years in a tertiary care hospital of Lahore. METHODS: An Analytical cross-sectional study was conducted in Pediatrics Outpatient Department at Akhtar Saeed Trust Teaching Hospital, Lahore from December 2017 to July 2018. Two hundred children of ages under five years coming to outdoor for treatment of minor ailments were included after informed consent from their parents. Non-probability, convenient sampling technique was used to collect sample. Data collected and analyzed on SPSS version 19. To find out association of stunting with multiple qualitative variables, chi-square test was applied and p-value was fixed at ≤ 0.05 to be significant. RESULTS: Out of 200 children screened in OPD, 42 (21.0%) were found to be stunted. The total percentage of stunting in male children was 28 (66.6%) and in female children were 14 (33.3%). Stunting was significantly associate with male gender (p=0.047), joint family system (p=0.049), low literacy level in mothers (p=0.031), unvaccinated status (p=0.003) and history of bottle feeding (p=0.037). CONCLUSION: The frequency of stunting in children less than five years of age is 42 (21.0%). The significant risk factors associated with stunting were found as male gender (p= 0.047), joint family system (p=0.049), low maternal education (p=0.031), unvaccinated status(p=0.03).

Mycoremediation: a treatment for heavy metal-polluted soil using indigenous metallotolerant fungi.

Bioleaching of heavy metals from industrial contaminated soil using metallotolerant fungi is the most efficient, cost-effective, and eco-friendly technique. In the current study, the contaminated soil samples from Hattar Industrial Estate revealed a total lead (Pb) and mercury (Hg) concentration of 170.90 mg L-1 and 26.66 mg L-1, respectively. Indigenous metallotolerant fungal strains including Aspergillus niger M1, Aspergillus fumigatus M3, Aspergillus terreus M6, and Aspergillus flavus M7 were isolated and identified by pheno- and genotyping. A. fumigatus and A. flavus of soil sample S1 showed higher efficiency for Pb removal (99.20% and 99.30%, respectively), in SDB medium. Likewise, A. niger and A. terreus of soil sample S2 showed higher efficiency for Hg removal (96% and 95.50%, respectively), in YPG medium. Furthermore, the maximum uptake efficiency for Pb removal (8.52 mg g-1) from soil sample S1 was noticed for A. fumigatus in YPG medium, while the highest uptake efficiency (4.23 mg g-1) of A. flavus M2 strain was observed with CYE medium. Similarly, the maximum uptake efficiency of 0.41 mg g-1 and 0.44 mg g-1 for Hg removal from soil sample S2 was found for A. niger and A. terreus strains, respectively, in CYE medium. Thus, in order to address the major issue of industrial waste pollution, indigenous fungal strains A. fumigatus (M1) and A. terreus (M7), isolated in this study, could be used (ex situ or in situ) to remediate soils contaminated with Pb and Hg.

Mycoremediation of heavy metal (Cd and Cr)-polluted soil through indigenous metallotolerant fungal isolates.

Remediation of heavy metals, other than microbial bioleaching method, is expensive and unsuitable for large contaminated areas. The current study was aimed to isolate, identify, and test the potential of indigenous fungal strains for heavy metal removal from contaminated soil. A total of three metallotolerant fungal strains, i.e., Aspergillus niger (M1DGR), Aspergillus fumigatus (M3Ai), and Penicillium rubens (M2Aii), were isolated and identified by phenotyping and genotyping from heavy metal-contaminated soil of  Hattar Industrial Estate, Pakistan. A. niger was found to be the most successful strain for the removal of heavy metals from the contaminated soil with maximum bioaccumulation efficiency of 98% (Cd) and 43% (Cr). In contrast, A. fumigatus showed comparatively low but still considerable bioleaching potential, i.e., 79% and 69% for Cd and Cr removal, respectively. Maximum metal uptake efficiency, i.e., 0.580 mg g-1 and 0.152 mg g-1 by A. niger strain was noticed for Cd and Cr with Czapek yeast extract (CYE) and Sabouraud dextrose broth (SDB) media, respectively. A. fumigatus (M3Ai) exhibited the maximum bioleaching capacity (0.40 mg g-1) for Cr with CYE medium. The results reveal that A. niger M1DGR and A. fumigatus M3Ai could be used to develop new strategies to remediate soil contaminated with heavy metals (Cd and Cr) through either in situ or ex situ mycoremediation.

CRISPR/Cas system: A game changing genome editing technology, to treat human genetic diseases.

Genes, are the functional units of heredity that used as an instructors to make proteins either to become the functional or structural part of the cell. Hence, the proteins get more attention because most of the life functions depends on it. Any mutation or alteration in genome sequences results in complete loss of function or formation of abnormal protein which leads to hereditary disorder. Gene therapy on the other hand, used as a remedy, a process that make correction in the gene which is responsible for genomic disorders. The treatment of disease state depends on the understanding of their genetic basis. While, numerous molecular genome editing tools have been developed and are being utilized to translate the abstract of gene therapy into reality, but the problem is still a mystery. The genome editing molecular scissors can be applied to dictate the selected genetic products that can have the therapeutic power. Thus, editing the specific sequences depends on the type of strategies being used by a molecule such is HDR or NHEJ. CRISPR/Cas9 editing technology can use in disease model to study the genitival disorders. One side the CRISPR technology seemed to be extremely accurate but on the other side it has some harmful effects i.e. Cas9 proteins sometimes cuts the similar sequences other than the specific targeted and Off-targeting Sequences etc. Urgent attention and improvement are needed for various implication of CRISPR/Cas9 technology, including the delivery, precision and control over the mention system. This review presents the current scenario of genome editing in vivo and its implications for the future of human genetic disease treatment as well as genome throughput potency.

Frequency distribution and risk factors of hepatitis B virus and hepatitis C virus infections among thalassemia patients: a regional study.

BACKGROUND: Thalassemia is a group of inherited hematological disorders caused by mutation in globin's genes. Regular blood transfusion lengthens the life of thalassemia patients but it carries a definite risk of the infections of blood-borne diseases. AIM/OBJECTIVE: The current study was carried out for the frequency distributions and risk factors of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections among thalassemia patients in Hazara regions, Pakistan. METHODS: A total of 324 enrolled thalassemia major patients were diagnosed in five different centers of Hazara regions. The study participants were screened for HBV and HCV using the immunochromatographic techniques test and real-time PCR for immunochromatographic technique-positive specimens. RESULTS: Out of the 324 major thalassemia patients, 24 (7.41%) were diagnosed with HBV and HCV infections. In total, 206 were male patients and the rate of HBV and HCV infections was 0.97% (two patients) and 3.88% (eight patients), respectively. Similarly, 118 were female patients and the rate of HBV-positive patients was 3.39% (four patients) and HCV was 8.47% (10 patients). The results also showed that 50% of HBV and HCV infections were found in the age group of 26-30 years, while 1.81% was found in the age group of 11-15 years. The positive HBV and HCV samples were also verified with the band size of 242 and 227 bp, respectively. CONCLUSION: Thus, to reduce the incidence of HBV and HCV in thalassemia patients, we must call for critical look on the transfusion practices as well as adoption of stricter donor selection.

Cloning and characterization of F3PYC gene encoding pyruvate carboxylase in Aspergillus flavus strain (F3).

Pyruvate carboxylase is a major enzyme for biosynthesis of organic acids like; citric acid, fumeric acid, and L-malic acid. These organic acids play very important role for biological remediation of heavy metals. In this study, gene walking method was used to clone and characterize pyruvate carboxylase gene (F3PYC) from heavy metal resistant indigenous fungal isolate Aspergillus flavus (F3). 3579 bp of an open reading frame which encodes 1193 amino acid protein (isoelectric point: 6.10) with a calculated molecular weight of 131.2008 kDa was characterized. Deduced protein showed 90-95% similarity to those deduced from PYC gene from different fungal strains including; Aspergillus parasiticus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus clavatus, and Aspergillus niger. Protein generated from the PYC gene was a homotetramer (α4) and having four potential N-linked glycosylation sites and had no signal peptide. Amongst most possible N-glycosylation sites were -N-S-S-I- at 36 amino acid, -N-G-T-V- at 237 amino acid, N-G-S-S- at 517 amino acid, and N-T-S-R- at 1111 amino acid, with several functions have been proposed for the carbohydrate moiety such as thermal stability, pH, and temperature optima for activity and stabilization of the three-dimensional structure. Hence, cloning of F3PYC gene from A. flavus has important biotechnological applications.

Cloning and characterization of pyruvate carboxylase gene responsible for calcium malate overproduction in Penicillium viticola 152 and its expression analysis.

In this study, a pyruvate carboxylase gene (PYC) from a marine fungus Penicillium viticola 152 isolated from marine algae was cloned and characterized by using Genome Walking method. An open reading frame (ORF) of The PYC gene (accession number: KM593097) had 3582bp encoding 1193 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 131.2757kDa. A putative promoter (intronless) of the gene was located at -666bp and contained a TATA box, several CAAT boxes, the 5'-SYGGRG-3' and a 5'-HGATAR-3' sequences. A consensus polyadenylation site (AATAAA) was also observed at +10bp downstream of the ORF. The protein deduced from the PYC gene had no signal peptide, was a homotetramer (4), and had the four functional domains. Furthermore, PYC protein also had three potential N-linked glycosylation sites, among them, -N-S-T-I- at 36 amino acid, -N-G-T-V- at 237 amino acid, and -N-G-S-S- at 517 amino acid were the most possible N-glycosylation sites. After expression of the PYC gene of P. viticola 152 in medium supplemented with CSL and biotin, it was found that the specific pyruvate carboxylase activity in MA production medium supplemented with CSL was much higher (0.5U/mg) than in MA medium supplemented with biotin (0.3U/mg), suggesting that optimal concentration of CSL is required for increased expression of the PYC gene, which is responsible for high level production of malic acid in P. viticola 152 strain.

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.