Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells.

Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX-the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.

Studies of the extracytoplasmic function sigma factor PG0162 in Porphyromonas gingivalis.

PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis, is composed of 193 amino acids. As previously reported, the PG0162-deficient mutant, P. gingivalis FLL350 showed significant reduction in gingipain activity compared with the parental strain. Because this ECF sigma factor could be involved in the virulence regulation in P. gingivalis, its genetic properties were further characterized. A 5'-RACE analysis showed that the start of transcription of the PG0162 gene occurred from a guanine (G) residue 69 nucleotides upstream of the ATG translation initiation codon. The function of PG0162 as a sigma factor was confirmed in a run-off in vitro transcription assay using the purified rPG0162 and RNAP core enzyme from Escherichia coli with the PG0162 promoter as template. As an appropriate PG0162 inducing environmental signal is unknown, a strain overexpressing the PG0162 gene designated P. gingivalis FLL391 was created. Compared with the wild-type strain, transcriptome analysis of P. gingivalis FLL391 showed that approximately 24% of the genome displayed altered gene expression (260 upregulated genes; 286 downregulated genes). Two other ECF sigma factors (PG0985 and PG1660) were upregulated more than two-fold. The autoregulation of PG0162 was confirmed with the binding of the rPG0162 protein to the PG0162 promoter in electrophoretic mobility shift assay. In addition, the rPG0162 protein also showed the ability to bind to the promoter region of two genes (PG0521 and PG1167) that were most upregulated in P. gingivalis FLL391. Taken together, our data suggest that PG0162 is a sigma factor that may play an important role in the virulence regulatory network in P. gingivalis.

VimA-dependent modulation of acetyl coenzyme A levels and lipid A biosynthesis can alter virulence in Porphyromonas gingivalis.

The Porphyromonas gingivalis VimA protein has multifunctional properties that can modulate several of its major virulence factors. To further characterize VimA, P. gingivalis FLL406 carrying an additional vimA gene and a vimA-defective mutant in a different P. gingivalis genetic background were evaluated. The vimA-defective mutant (FLL451) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimA-defective mutant (FLL92) in the P. gingivalis W83 genetic background. In contrast to the wild type, gingipain activity was increased in P. gingivalis FLL406, a vimA chimeric strain. P. gingivalis FLL451 had a five times higher biofilm-forming capacity than the parent strain. HeLa cells incubated with P. gingivalis FLL92 showed a decrease in invasion, in contrast to P. gingivalis FLL451 and FLL406, which showed increases of 30 and 40%, respectively. VimA mediated coenzyme A (CoA) transfer to isoleucine and reduced branched-chain amino acid metabolism. The lipid A content and associated proteins were altered in the vimA-defective mutants. The VimA chimera interacted with several proteins which were found to have an LXXTG motif, similar to the sorting motif of gram-positive organisms. All the proteins had an N-terminal signal sequence with a putative sorting signal of L(P/T/S)X(T/N/D)G and two unique signatures of EXGXTX and HISXXGXG, in addition to a polar tail. Taken together, these observations further confirm the multifunctional role of VimA in modulating virulence possibly through its involvement in acetyl-CoA transfer and lipid A synthesis and possibly by protein sorting.

Field screening and evaluation of pigeonpea genotypes against podborer (Helicoverpa armigera).

Hence an experiment was conducted to screen fifteen germplasm lines for their resistance/tolerance to podborer under natural infestation in pesticide-free open field. To asses the degree of infestation 250 green pods were randomly picked up from all the plants of each plot. The pods damaged by gram podborer were detected by the presence large bored holes in pods. Such infested and healthy pods were counted separately. The pod damage (%) was calculated on the basis of number of pods examined and the number of infested pods. Marketable green pod yield of each genotype under natural pest infestation was also recorded. On the basis of mean infestation, ICP13201 showed the lowest (25%) pod damage and showed lowest susceptibility among the genotypes studied. It was followed by ICP13208 and ICP11964 showed lower pod damage. The rest of the genotype suffered higher pod damage to Helicoverpa armigera. Considering the yield potential ICP13201, ICP13214 and ICP13212 showed higher yield potential than other genotypes. Considering the lower susceptibility to Helicoverpa armigera and higher yield potential ICP13201 was found to be the best.

Uptake of Tc-99m sestamibi by brown tumors in a patient with primary hyperparathyroidism.

This article discusses a case of Tc-99m sestamibi uptake by brown tumors in a patient with primary hyperparathyroidism. Brown tumors are characteristic tumors of hyperparathyroidism and often have well-defined margins. In this patient with clinical findings of hyperparathyroidism, a retrosternal parathyroid adenoma was identified using Tc-99m sestamibi parathyroid scintigraphy. The adenoma was resected surgically, and the diagnosis was confirmed histopathologically.

Fertility estimation from retrospective surveys: biases attributable to pregnancy-related movement of mothers.

In a number of developing countries, especially in South Asia, there is a custom for a pregnant woman to go to her mother's home for delivery and remain for some months afterwards. In this context, estimates of various fertility measures, based on data from a sample survey of resident women, will be seriously biased. Inclusion of data for visitors to the sample households does not fully compensate for this bias. The presence and magnitude of the bias is illustrated by the analysis of data from large-scale sample surveys conducted in the state of Orissa in India and by World Fertility Survey data from Bangladesh and Nepal.

Life table analyses of amenorrhoea and lactation and their relationship in a rural south Indian population.

PIP: This report attempts to provide estimates of the length of amenorrhea and lactations for a rural population based on data from a prospective survey and by using life table techniques. Amenorrhea is studied in relation to age, partiy, nature of pregnancy termination, and lactation duration in married women for possible differentials. To study the relationship between lactation and amenorrheaa prospective Standard Fertility Survey with a stratified random sample of about 3000 households was carried out in Athoor Block, Madurai District, Tamil Nadu, by the Gandhigram Institute of Rural Health and Family Planning. The study results indicate, from the analysis of women whose lactation is shorter than their amenorrhea length, that lactation has a strong influence on the length of menstruation; no matter how many months lactation continues, the additional mean months of amenorrhea after cessation are usually 2-3. This result is supplemented by the fact that women who lactate longer have longer amenorrhea than those with shorter lactation. The latter result may be a little biased because in this analysis lactation during menstruating interval also is used. The relationship of age to amenorrhea lenth is positive. When women lactate a year or more, there could be an increase of 1 to 2 months of amenorrhoea from younger to older women. Thus, the older women who have lengthy amenorrhea have more chances of being omitted in this analysis because of the study design and so the increase in amenorrhea length by age is understated. It would be inferred then that the effect of age on amenorrhea length could be more than indicated.^ieng