Emergent chemodiversity: The case of stereoisomerism in acyclic alkanes

The objective of this pen‐and‐paper study is to witness the emergence of stereoisomeric properties when comparing lower to higher families of homologs. Specifically, the study compares all acyclic hexanes (five constitutional isomers, a.k.a. regioisomers), all nine heptanes, all 18 octanes, all 35 nonanes, and all 75 decanes. The first part of the work examines the nature and number of stereoisomeric properties seen to emerge in chemical structures featuring one chiral center (i.e., enantiomerism) or two such centers, in which case more complex stereoisomeric features emerge (enantiomerism, diastereoisomerism, pseudoasymmetry, and meso ‐isomers). The first emergence of chirality (i.e., enantiomerism) occurred in some heptanes. Diastereoisomerism and meso ‐isomers appear with some octanes, while a pseudoasymmetric center exists in a decane regioisomer. The second part of the work is an attempt to rationalize the numbers of regioisomers, chiral centers, and stereoisomers as these numbers grow from one family of regioisomers to the higher ones. Far from being random, such increases prove regular and ordered.     

Functionalities tuned enantioselectivity of phenylcarbamate cyclodextrin clicked chiral stationary phases in HPLC

The mixed chloro‐ and methyl‐ functionalities can greatly modulate the enantioselectivities of phenylcarbamate cyclodextrin (CD) clicked chiral stationary phases (CSPs). A comparison study is herein reported for per(4‐chloro‐3‐methyl)phenylcarbamate and per(2‐chloro‐5‐methyl)phenylcarbamate β‐CD clicked CSPs (i.e., CCC4M3‐CSP and CCC2M5‐CSP). The enantioselectivity dependence on column temperature was studied in both normal‐phase and reversed‐phase mode high performance liquid chromatography (HPLC). The thermodynamic study revealed that the stronger intermolecular interactions can be formed between CCC4M3‐CSP and chiral solutes to drive the chiral separation. The higher enantioselectivities of CCC4M3‐CSP were further demonstrated with the enantioseparation of 17 model racemates in HPLC.     

Isolation,Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson’s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.     

Isolation and partial characterization of 3 nontoxic d‐galactose–specific isolectins from seeds of Momordica balsamina

Three isolectins denoted hereforth MBaL‐30, MBaL‐60, and MBaL‐80 were isolated from seeds extract of Momordica balsamina by 30%, 60%, and 80% ammonium sulfate saturations, respectively. The native molecular weights of these lectins, as judged by gel filtration, were 108, 56, and 160 kDa, respectively. On SDS‐PAGE, under reduced condition, 27 kDa band was obtained for all isolectins. The lectins hemagglutinating activities were variably inhibited by d ‐galactose (minimum inhibitory concentrations = 12.5mM, 50mM, and 0.391mM, respectively). MBaL‐30 and ‐60 could agglutinate all human blood types with slight preference for the A and O blood groups, whereas MBaL‐80 did not agglutinate B and AB blood types. The 3 isolectins were purified from crude seeds extract, collectively, in a single step on the affinity matrix Lactamyl‐Seralose 4B; this purified lectin fraction, which contains all isolectins, is termed MBaL. The N‐terminal of MBaL till the 25th amino acid was NLSLSELDFSADTYKSFIKNLRKQL, which shares 88% sequence identity with Momordica charantia lectin type‐2 ribosomal inactivating protein from Momordica charantia and 50% with momordin II from Momordica balsamina . MBaL retained 100% activity at up to 50°C for 30 minutes. MBaL‐30 and MBaL‐60 exhibited maximum activities in the pH range between 4 and 8, while MBaL‐80 was showing maximum activity in the pH range between 3 and 5. Treatment of MBaL‐30 and MBaL‐60 with EDTA completely abolished their hemagglutinating activities. Addition of Zn and Fe ions to the ethylenediaminetetraacetic acid–treated MBaL‐30 and MBaL‐60 lectins did not only regained the loss of activity but also resulted in 200% to 300% increase in activity, respectively. MBaL‐30 and ‐60 agglutinated gram positive Listeria monocytogenes and Staphylococcus aureus, whereas MBaL‐30 could merely agglutinate Escherichia coli . None of these lectins could arrest bacterial growth. Addition of MBaL to cancer cell lines (Gastric cancer cell line (AGS) and Gastric cencer cell line (MKN45), Glioblastoma (ECV‐304), and Human urinary bladder cancer cell line (U87‐MG)) at varying concentrations did not cause statistically significant changes on cell growth and viability.     

Methylation of KvDMR1 involved in regulating the imprinting of CDKN1C gene in cattle

The CDKN1C gene encodes a cyclin‐dependent kinase inhibitor and is one of the key genes involved in the development of Beckwith–Wiedemann syndrome and cancer. In this study, using a direct sequencing approach based on a single nucleotide polymorphism (SNP) at genomic DNA and cDNA levels, we show that CDKN1C exhibits monoallelic expression in all seven studied organs (heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) in cattle. To investigate how methylation regulates imprinting of CDKN1C in cattle, allele‐specific methylation patterns in two putative differential methylation regions (DMRs), the CDKN1C DMR and KvDMR1, were analyzed in three tissues (liver, spleen and lung) using bisulfite sequencing PCR. Our results show that in the CDKN1C DMR both parental alleles were unmethylated in all three analyzed tissues. In contrast, KvDMR1 was differentially methylated between the two parental alleles in the same tissues. Statistical analysis showed that there is a significant difference in the methylation level between the two parental alleles (P < 0.01), confirming that this region is the DMR of KvDMR1 and that it may be correlated with CDKN1C imprinting.     

In Vitro Cultivation of Borrelia duttonii on Cultures of SflEp Cells

Borrelia duttonii strain 406 K, a causative agent of relapsing fever, could not be cultivated in vitro in currently available media for borreliae. We have developed an in vitro cultivation system by using SflEp cell cultures. The average increases of the number of borreliae, when inoculated with 1.0 × 10⁵ organisms per ml from infected mice, were 23-fold and 150-fold in the primary culture and the 3rd subculture, respectively. Even a single borrelia could propagate in this cultivation system. This system will be useful for immunological and physiological studies on uncultivable Borrelia strains.