Mutations in the Alpha 1,2-Mannosidase Gene, MAN1B1, Cause Autosomal-Recessive Intellectual Disability

We have used genome-wide genotyping to identify an overlapping homozygosity-by-descent locus on chromosome 9q34.3 (MRT15) in four consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability (NS-ARID) and one in which the patients show additional clinical features. Four of the families are from Pakistan, and one is from Iran. Using a combination of next-generation sequencing and Sanger sequencing, we have identified mutations in the gene MAN1B1, encoding a mannosyl oligosaccharide, alpha 1,2-mannosidase. In one Pakistani family, MR43, a homozygous nonsense mutation (RefSeq number {"type":"entrez-nucleotide","attrs":{"text":"NM_016219.3","term_id":"218749882","term_text":"NM_016219.3"}}NM_016219.3: c.1418G>A [p.Trp473^∗]), segregated with intellectual disability and additional dysmorphic features. We also identified the missense mutation c. 1189G>A (p.Glu397Lys; RefSeq number {"type":"entrez-nucleotide","attrs":{"text":"NM_016219.3","term_id":"218749882","term_text":"NM_016219.3"}}NM_016219.3), which segregates with NS-ARID in three families who come from the same village and probably have shared inheritance. In the Iranian family, the missense mutation c.1000C>T (p.Arg334Cys; RefSeq number {"type":"entrez-nucleotide","attrs":{"text":"NM_016219.3","term_id":"218749882","term_text":"NM_016219.3"}}NM_016219.3) also segregates with NS-ARID. Both missense mutations are at amino acid residues that are conserved across the animal kingdom, and they either reduce k_cat by ∼1300-fold or disrupt stable protein expression in mammalian cells. MAN1B1 is one of the few NS-ARID genes with an elevated mutation frequency in patients with NS-ARID from different populations.     

Mixed Convective Peristaltic Flow of Water Based Nanofluids with Joule Heating and Convective Boundary Conditions

Main objective of present study is to analyze the mixed convective peristaltic transport of water based nanofluids using five different nanoparticles i.e. (Al₂O_3, CuO, Cu, Ag and TiO₂). Two thermal conductivity models namely the Maxwell''s and Hamilton-Crosser''s are used in this study. Hall and Joule heating effects are also given consideration. Convection boundary conditions are employed. Furthermore, viscous dissipation and heat generation/absorption are used to model the energy equation. Problem is simplified by employing lubrication approach. System of equations are solved numerically. Influence of pertinent parameters on the velocity and temperature are discussed. Also the heat transfer rate at the wall is observed for considered five nanofluids using the two phase models via graphs.     

l-tryptophan-assisted PGPR-mediated induction of drought tolerance in maize (Zea mays L.)

Drought is an important abiotic stress that limits the plant growth and productivity. Present investigation was aimed that plant growth-promoting rhizobacteria (PGPR) isolated from moisture-stressed area impart drought tolerance in plants and tryptophan may improve their efficiency. Pseudomonas sp. (1), Bacillus cereus and Bacillus pumilus (B. pumilus) were isolated from maize rhizosphere grown in irrigated fields, semi-arid region and arid region, respectively. Proteus sp. and Pseudomonas sp. (2) were isolated from rice rhizosphere grown in irrigated fields and raised bed. B. pumilus produced 5× more abscisic acid (ABA) in culture media than Pseudomonas sp. (1) by the addition of l-tryptophan. These inoculants also modulated the phytohormone content of maize leaves in a pot experiment. Higher ABA was produced by the application of B. pumilus and Pseudomonas sp. (2), while indole 3-acetic acid and gibberellic acid were found higher in Pseudomonas sp. (1) and Proteus sp. treated plants. Addition of l-tryptophan increased the concentration of all phytohormones in soil and leaves of maize. Maximum increase in relative water content, osmotic potential, protein content and photosynthetic pigments was recorded in B. pumilus treated maize plants. Under irrigated condition, response of Pseudomonas sp. co-inoculated with B. pumilus from arid field superseded while under drought stress the effect of later predominated. Bacillus pumilus can be used in the formulation of biofertilizer to alleviate drought stress in arid and semi-arid regions.     

Remarkable sequence conservation of the last intron in the PKD1 gene

The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation.     

Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor

For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O₂ concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 10⁵ cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O₂ uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.     

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps.     

Ocaratuzumab,an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab’s potency against CLL cells. In vitro assessment of ocaratuzumab’s direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P < 0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1–10 ug/ml; P < 0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P < 0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.     

A new species ofPharoscymnus Bedel [Coleoptera: Coccinellidae] predaceous on scale insects in Pakistan

During the course of studies on scale insects and their natural enemies in West Pakistan, this species was found to be predaceous onAonidiella orientalis (Newst.),Chrysomphalus ficus (Ashm.),Parlatoria blanchardii (Targ.),P. crypta Mck. andTecaspis sp. Its population was extremely low except at Mirpur Khas (Sub-coastal area) where it was abundant onA. orientalis attackingMusa sapientum. It proved to be new to science and is described below.     

Improving Quantitative and Qualitative Characteristics of Wheat (<i>Triticum aestivum</i> L.) through Nitrogen Application under Semiarid Conditions

Nitrogen (N), the building block of plant proteins and enzymes, is an essential macronutrient for plant functions. A field experiment was conducted to investigate the impact of different N application rates (28, 57, 85, 114, 142, 171, and 200 kg ha⁻¹) on the performance of spring wheat (cv. Ujala-2016) during the 2017–2018 and 2018–2019 growing seasons. A control without N application was kept for comparison. Two years mean data showed optimum seed yield (5,461.3 kg ha⁻¹) for N-application at 142 kg ha⁻¹ whereas application of lower and higher rates of N did not result in significant and economically higher seed yield. A higher seed yield was obtained in the 2017–2018 (5,595 kg ha⁻¹) than in the 2018–2019 (5,328 kg ha⁻¹) growing seasons under an N application of 142 kg ha⁻¹. It was attributed to the greater number of growing degree days in the first (1,942.35°C days) than in the second year (1,813.75°C). Higher rates of N (171 and 200 kg ha⁻¹) than 142 kg ha⁻¹ produced more number of tillers (i.e., 948,300 and 666,650 ha⁻¹, respectively). However, this increase did not contribute in achieving higher yields. Application of 142, 171, and 200 kg ha⁻¹ resulted in 14.15%, 15.0% and 15.35% grain protein concentrations in comparison to 13.15% with the application of 114 kg ha⁻¹. It is concluded that the application of N at 142 kg ha⁻¹ could be beneficial for attaining higher grain yields and protein concentrations of wheat cultivar Ujala-2016.

     

Study of the structure and properties of native and hydrothermally processed wild-type, lam and r variant pea starches that affect amylolysis of these starches

Starches from WT, lam, and r pea mutants differing in amylopectin/amylose contents (70, 90, and 28% amylopectin, respectively) were used in kinetic studies of pancreatic α-amylase action at 37 °C and for investigations of their supramolecular structure and physicochemical properties during heating. For WT and lam starches, amylase accessibility and catalytic efficiencies (CE) increased following hydrothermal processing up to 100 °C. Accessibility changed relatively less in r during heating with increasing K(m) between 60-90 °C. Limiting values of K(m) after gelatinization were very similar for all three mutants, indicating that relative proportions of amylose/amylopectin have little influence on amylase accessibility once ordered structures are lost. For WT and lam, increases in enzyme accessibility and CE paralleled a rise in amorphous content. It is suggested that the complex behavior for r resulted from amylose gel formation between 60-90 °C. Amorphous amylopectin seems a better substrate for amylase than amorphous amylose.