Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates

Biocatalysis has been increasingly used for pharmaceutical synthesis in an effort to make manufacturing processes greener and more sustainable. Biocatalysts that possess excellent activity, specificity, thermostability and solvent-tolerance are highly sought after to meet the requirements of practical applications. Generating biocatalysts with these specific properties can be achieved by either discovery of novel biocatalysts or protein engineering. Meanwhile, chemoenzymatic routes have also been designed and developed for pharmaceutical synthesis on an industrial scale. This review discusses the recent discoveries, engineering, and applications of biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. Key classes of biocatalysts include reductases, oxidases, hydrolases, lyases, isomerases, and transaminases.     

Effects of caffeine on in vivo and in vitro oocyte maturation in mice

The objective was to investigate, using a mouse model, the effects of caffeine on the number of ovulated oocytes, the rate of oocyte maturation, the susceptibility of oocytes to activating stimuli, spindle morphology, and distribution of cortical granules (CGs). Mice were given caffeine (150 mg/kg body weight ip) at various times relative to hCG (-2, 0, and +2h); in an in vitro study, 1, 5 or 10 mM caffeine was added to the maturation culture. Caffeine had no effect on the quality of oocytes in vivo maturation, but caffeine was detrimental to the quality of oocytes matured in vitro. Further studies are needed to determine caffeine concentration in follicles relative to that in culture medium.     

Three-dimensional reconstruction of brain surface anatomy based on magnetic resonance imaging diffusion-weighted imaging: A new approach

Fifty normal noninfarct patients and 12 cases with infarcts of the cerebrum were examined with routine magnetic resonance imaging and echo-planar diffusion-weighted imaging. The diffusion-weighted three-dimensional images were reconstructed with volume-rendering processing on workstation. Precentral gyrus, post-central gyrus, superior parietal lobule, superior frontal gyrus, precentral sulcus, central sulcus, postcentral sulcus, intraparietal sulcus and superior frontal sulcus were best shown of all structures with an arbitrary score of 2.61–2.77. Supramarginal gyrus, middle frontal gyrus, inferior frontal gyrus and lateral sulcus were clearly shown in the majority of the cerebra with average scores of 2.0–2.49; angular gyrus, inferior frontal sulcus and superior temporal gyrus were not demonstrated satisfactorily and their average scores were 1.67–1.89. Middle temporal gyrus, inferior temporal gyrus, superior temporal sulcus and inferior temporal sulcus were difficult to identify, and thus had average scores of 0.87–1.26. Brain surface structures were better displayed in the older group of individuals than in the younger group. The structures in the 12 cases with acute or chronic cerebrum infarcts were also satisfactorily demonstrated with this new technique.     

Expression Profiling and Validation of Potential Reference Genes During <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> Embryogenesis

Differential expression of genes is crucial to embryogenesis. The analysis of gene expression requires appropriate references that should be minimally regulated during the embryonic development. To select the most stable genes for gene normalization, the expression profiles of eight commonly used reference genes (ACTB, GAPDH, rpL17, α-Tub, EF1-α, UbcE, B2M, and 18S rRNA) were examined during Japanese flounder (Paralichthys olivaceus) embryonic development using quantitative real-time polymerase chain reaction. It was found that all seven mRNA genes appeared to be developmentally regulated and exhibited significant variation of expression. However, further analyses revealed the stage-specific expression stability. Hence when normalization using these mRNA genes, the differential and stage-related expression should be considered. 18S rRNA gene, on the other hand, showed the most stable expression and could be recommended as a suitable reference gene during all embryonic developmental stages in P. olivaceus. In summary, our results provided not only the appropriate reference gene for embryonic development research in P. olivaceus, but also possible guidance to reference gene selection for embryonic gene expression analyses in other fish species.     

Contrasting Patterns in the Early Stage of SARS-CoV-2 Evolution between Humans and Minks

One of the unique features of SARS-CoV-2 is its apparent neutral evolution during the early pandemic (before February 2020). This contrasts with the preceding SARS-CoV epidemics, where viruses evolved adaptively. SARS-CoV-2 may exhibit a unique or adaptive feature which deviates from other coronaviruses. Alternatively, the virus may have been cryptically circulating in humans for a sufficient time to have acquired adaptive changes before the onset of the current pandemic. To test the scenarios above, we analyzed the SARS-CoV-2 sequences from minks (Neovision vision) and parental humans. In the early phase of the mink epidemic (April to May 2020), nonsynonymous to synonymous mutation ratio per site in the spike protein is 2.93, indicating a selection process favoring adaptive amino acid changes. Mutations in the spike protein were concentrated within its receptor-binding domain and receptor-binding motif. An excess of high-frequency derived variants produced by genetic hitchhiking was found during the middle (June to July 2020) and late phase I (August to September 2020) of the mink epidemic. In contrast, the site frequency spectra of early SARS-CoV-2 in humans only show an excess of low-frequency mutations, consistent with the recent outbreak of the virus. Strong positive selection in the mink SARS-CoV-2 implies that the virus may not be preadapted to a wide range of hosts and illustrates how a virus evolves to establish a continuous infection in a new host. Therefore, the lack of positive selection signal during the early pandemic in humans deserves further investigation.     

Southern Ocean Biogeography of Tintinnid Ciliates of the Marine Plankton

Ciliate microzooplankton are important grazers in most pelagic ecosystems and among them, tintinnids, with their largely species‐specific loricas, allow relatively easy assessment of questions of diversity and distributions. Herein, we present the results of a survey of species records of tintinnids from the Southern Ocean (locations below 40°S) reported in 56 publications yielding 2,047 species records (synonyms included) from 402 locations. The 192 species reported can be parsed into two main groups: 32 endemic Southern Ocean species, known only from 40°S and further south, and a second group of 181 widespread species, forms with extensive geographic ranges extending into the Southern Ocean. Widespread species reported from the Southern Ocean can be further divided into a group of 81 species, each recorded multiple times in the Southern Ocean waters and 70 apparent “stray” species which have only been found but once. The endemic and widespread species of the Southern Ocean show both distinct distributional patterns and morphological differences. The assemblage of Southern Ocean endemics is found mostly within the Antarctic zone delimited by the average location of the Polar Front and contains a relatively large portion of wide‐mouthed forms. We give suggestions for future study.     

Natural Killer Cells Are Characterized by the Concomitantly Increased Interferon-γ and Cytotoxicity in Acute Resolved Hepatitis B Patients

Natural killer (NK) cells are abundant in the liver and have been implicated in inducing hepatocellular damage in patients with chronic hepatitis B virus (HBV) infection. However, the role of NK cells in acute HBV infection remains to be elucidated. We comprehensively characterized NK cells and investigated their roles in HBV clearance and liver pathology in 19 chronic hepatitis B (CHB) patients and 21 acute hepatitis B (AHB) patients as well as 16 healthy subjects. It was found that NKp46⁺ NK cells were enriched in the livers of AHB and CHB patients. We further found that peripheral NK cells from AHB patients expressed higher levels of activation receptors and lower levels of inhibitory receptors than those from CHB patients and HC subjects, thus displaying the increased cytolytic activity and interferon-γ production. NK cell activation levels were also correlated positively with serum alanine aminotransferase levels and negatively with plasma HBV DNA levels in AHB patients, which is further confirmed by the longitudinal follow-up of AHB patients. Serum pro-inflammatory cytokine and chemokine levels were also increased in AHB patients as compared with CHB and HC subjects. Thus, the concomitantly increased interferon-γ and cytotoxicity of NK cells were associated with liver injury and viral clearance in AHB patients.     

Effects of MTNR1B variants on fasting glucose levels in a Korean population

Fasting glucose level is the most basic and widely used indicator of diabetes. Several genome wide association studies have reported that the gene encoding melatonin receptor 1B (MTNR1B) exerts a major effect on serum fasting glucose levels. We tested for the association between single nucleotide polymorphisms (SNPs) in the MTNR1B gene and fasting glucose levels in a Korean population consisting of 8,229 subjects taken from two community-based cohorts. The mean age of the subjects in the study population was 51.9 years. For this study, we selected 363 SNPs located in the MTNR1B gene, which is located on chromosome 11. Multivariate linear regression models were used to test for genotypic effects on fasting glucose levels while adjusting for age and sex under an additive model. The MTNR1B SNP most highly associated with fasting glucose levels was rs10830962 (p=1.95×10^?5), followed by rs3847554 (p=3.16×10^?4). Replication of these initial findings is important to better understand the correlation between MTNR1B variations and their effects, especially in Asian populations.     

atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain

Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor organs. Here, we show that ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function, regulating arborization during larval and pupal development by interacting with Notch.     

Mechanochemical regulation of oscillatory follicle cell dynamics in the developing Drosophila egg chamber

During tissue elongation from stage 9 to stage 10 in Drosophila oogenesis, the egg chamber increases in length by ∼1.7-fold while increasing in volume by eightfold. During these stages, spontaneous oscillations in the contraction of cell basal surfaces develop in a subset of follicle cells. This patterned activity is required for elongation of the egg chamber; however, the mechanisms generating the spatiotemporal pattern have been unclear. Here we use a combination of quantitative modeling and experimental perturbation to show that mechanochemical interactions are sufficient to generate oscillations of myosin contractile activity in the observed spatiotemporal pattern. We propose that follicle cells in the epithelial layer contract against pressure in the expanding egg chamber. As tension in the epithelial layer increases, Rho kinase signaling activates myosin assembly and contraction. The activation process is cooperative, leading to a limit cycle in the myosin dynamics. Our model produces asynchronous oscillations in follicle cell area and myosin content, consistent with experimental observations. In addition, we test the prediction that removal of the basal lamina will increase the average oscillation period. The model demonstrates that in principle, mechanochemical interactions are sufficient to drive patterning and morphogenesis, independent of patterned gene expression.