Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be “disease causing,” with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Explicit solvation thermodynamics in ionic solution: extending grid inhomogeneous solvation theory to solvation free energy of salt–water mixtures

Hydration thermodynamics play a fundamental role in fields ranging from the pharmaceutical industry to environmental research. Numerous methods exist to predict solvation thermodynamics of compounds ranging from small molecules to large biomolecules. Arguably the most precise methods are those based on molecular dynamics (MD) simulations in explicit solvent. One theory that has seen increased use is inhomogeneous solvation theory (IST). However, while many applications require accurate description of salt–water mixtures, no implementation of IST is currently able to estimate solvation properties involving more than one solvent species. Here, we present an extension to grid inhomogeneous solvation theory (GIST) that can take salt contributions into account. At the example of carbazole in 1 M NaCl solution, we compute the solvation energy as well as first and second order entropies. While the effect of the first order ion entropy is small, both the water–water and water–ion entropies contribute strongly. We show that the water–ion entropies are efficiently approximated using the Kirkwood superposition approximation. However, this approach cannot be applied to the water–water entropy. Furthermore, we test the quantitative validity of our method by computing salting-out coefficients and comparing them to experimental data. We find a good correlation to experimental salting-out constants, while the absolute values are overpredicted due to the approximate second order entropy. Since ions are frequently used in MD, either to neutralize the system or as a part of the investigated process, our method greatly extends the applicability of GIST. The use-cases range from biopharmaceuticals, where many assays require high salt concentrations, to environmental research, where solubility in sea water is important to model the fate of organic substances.

The Role of Cations in Resorcinol–Formaldehyde Gel Textural Characteristics

The production of resorcinol–formaldehyde xerogels has yielded insight into the gelation processes underpinning their structures. In this work, the role of the cation species from the catalyst is probed by studying the simultaneous addition of sodium carbonate and calcium carbonate to a resorcinol–formaldehyde mixture. Twenty-eight xerogels were prepared by varying the solids content, catalyst concentration, and catalyst composition, and each was analysed for its textural properties, including the surface area and average pore diameter. The results indicate that the role of the cation is linked to the stabilisation of the clusters formed within the system, and that the Group II catalyst causes the salting out of the oligomers, resulting in fewer, larger clusters, hence, an increase in pore size and a broadening of the pore size distribution. The results provide insight into how these systems can be further controlled to create tailored porous materials for a range of applications.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by-products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U⋅mg-1. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

Purification of quantum dot-based bioprobes with a salting out strategy

A salting out strategy is reported for purification of IgG-conjugated QD (IgG-QD) bioprobes. The optical properties, target recognition, and colloidal stability of the purified IgG-QD were commendably maintained after salting out.

Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766)

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Pembuatan Konsentrat Protein Ampas Kelapa (Cocos nucifera L.) Bebas Lemak pada Berbagai Konsentrasi NaOH

Coconut pulp contains up to 18.2% protein which is one of the wastes from coconut oil processing. Protein from coconut pulp can be used in the manufacture of protein concentrates which are widely used in the food industry. The purpose of this research is to determine the concentration of NaOH that can produce the yield and protein content of coconut pulp protein concentrate. Coconut pulp protein was extracted with NaOH at various concentrations of 0.5, 1, 1.5, 2, and 2.5 M, then continued with salting out using salt (NH4)2SO4 with a saturation of 65%. The crude protein content of coconut pulp protein concentrate was determined by the Kjeldahl method. The results showed that the use of 0.5 M NaOH resulted in the highest protein content of coconut pulp protein concentrate, which was 71.30% with a yield of 14.42%.

Development of soapstock processing technology to ensure waste-free and safe production

Soapstock is a large-tonnage waste of the oil and fat industry, the disposal of which is environmentally hazardous. Processing of soapstock into industrially valuable products, in particular, fatty acids, is promising. The method for producing fatty acids, which consists in sequential saponification of soapstock with sodium hydroxide solution, salting out with sodium chloride and decomposition with sulfuric acid solution has been investigated. The feature of this work is the study of the effect of salting out conditions of saponified soapstock on the yield and neutralization number of fatty acids. As an experimental sample, sunflower soapstock was used, the indicators of which correspond to DSTU 5033 (CAS 68952-95-4): mass fraction of total fat – 67.3 %, fatty acids – 61.8 %, neutral fat – 5.5 %. Soapstock was subjected to preliminary saponification under the following conditions: duration 85 min., concentration of sodium hydroxide solution 45 %. After that, the saponified mass was subjected to salting out. The obtained core soap was decomposed with the sulfuric acid solution under the following conditions: temperature 90 °C, duration 40 min. Rational salting out conditions were determined: duration (80 min.) and sodium chloride concentration (16%). Under these conditions, the fatty acid yield is 95.0 %, the neutralization number is 194.8 mg KOH/g. The resulting fatty acids comply with DSTU 4860 (CAS 61788-66-7): the mass fraction of moisture and volatiles is 0.85 %, the mass fraction of total fat is 98.9 %, cleavage depth is 94.2 % oleic acid. This method of soapstock processing increases the fatty acid yield by 3.5 % compared to the method with saponification and decomposition, by 20.3 % compared to the method of soapstock decomposition with sulfuric acid. At the same time, the neutralization number increases by 4.1 % and 8.2 %, respectively. The improved method for fatty acids producing from soapstock provides high- quality fatty acids with increased yield.