Suneel Kumar Onteru

Curcumin Encapsulated in Milk Exosomes Resists Human Digestion and Possesses Enhanced Intestinal Permeability in Vitro

Exosomes, the extracellular secretary nano-vesicles, act as carriers of biomolecules to the target cells. They exhibit several attributes of an efficient drug delivery system. Curcumin, despite having numerous bioactive and therapeutic properties, has limited pharmaceutical use due to its poor water solubility, stability, and low systemic bioavailability. Hence, this study aims to enhance the therapeutic potential of curcumin, a model hydrophobic drug, by its encapsulation into milk exosomes. In the present study, we investigated the stability of free curcumin and exosomal curcumin in PBS and in vitro digestive processes. Additionally, their uptake and trans-epithelial transport were studied on Caco-2 cells. Curcumin in milk exosomes had higher stability in PBS, sustained harsh digestive processes, and crossed the intestinal barrier than free curcumin. In conclusion, the encapsulation of curcumin into the exosomes enhances its stability, solubility, and bioavailability. Therefore, the present study demonstrated that milk exosomes act as stable oral drug delivery vehicles.

Curcumin primed exosomes reverses LPS‐induced pro‐inflammatory gene expression in buffalo granulosa cells

Curcumin possesses anti‐inflammatory properties and provides a promising treatment for inflammation. The aim of the study is to establish that buffalo granulosa cells when primed with curcumin (20 μM), release improved cellular contents through exosome that can mitigate granulosa cell dysfunction. Recently, we have shown that buffalo granulosa cells exposed to LPS (1 μg/mL) in serum free culture, transiently increased the pro‐inflammatory cytokine genes (IL‐1β, TNF‐α, IL‐6) expression followed by the inhibition of CYP19A1 gene expression and estradiol production. Therefore, LPS‐treated granulosa cells were used as a model of inflammation and curcumin primed exosomes were utilized to check their potential for reducing granulosa cell dysfunction. Expression level of pro‐inflammatory cytokines and CYP19A1 were detected by real time PCR while estradiol levels were measured by ELISA. Exosomes derived from curcumin‐treated cells alleviated LPS mediated inflammation. In conclusion, our study potentiates the use of curcumin primed exosomes in mitigating granulosa cell dysfunction. Results show the therapeutic conservatories of curcumin via primed exosomes.

Exploration of Luteinizing hormone in murrah buffalo (Bubalus bubalis) urine: Extended surge window opens door for estrus prediction

Estrus detection in buffaloes has been a major concern for decades, and lack of reliable methods affects their effective reproductive management. Luteinizing hormone (LH) detection in urine is in practice for several mammals for timed insemination, whereas very few reports are available on buffalo urinary LH. The focus of this study is to detect the presence of LH in buffalo urine, quantitate variation in urinary LH during different estrous cycle phases and examine the duration of mid-cycle LH window. Nearly hundred buffaloes were examined, longitudinal urine samples (n=42) were collected from seventeen animals and classified into respective phases based on several estrus detection parameters. The urinary LH was detected using bovine LH ELISA kit validated for serum/plasma/tissue homogenate. Detection of buffalo LH in the neat urine convincingly proved the competence of the bovine LH kit. Variation in the LH range was observed between different phases of estrous cycle and significant fold variation (P<0.05) was noticed during estrus phase (1.01±0.23) with average baseline value of 46.73±3.36mIU/mL. Interestingly, an extended window (A1-A3) of mid-cycle LH surge was observed due to its lingering excretion in urine. The results, altogether, revealed that LH can be detected in buffalo urine with noticeable fold variation during estrus phase and the extended LH window intensifies the chance of ovulation prediction for timed insemination.

Small Interfering RNA in Milk Exosomes Is Resistant to Digestion and Crosses the Intestinal Barrier In Vitro

Milk is not only a composite of nutrients but emerged as a source of exosomes acting as a promising drug delivery vehicle for small interfering RNA (siRNA). siRNA is known for its immense therapeutic potential but has various physiological limitations, including stable delivery. To investigate the suitability of siRNA for physiological stability and oral delivery, we encapsulated scrambled Alexa Fluor (AF)-488 siRNA in milk whey exosomes using lipofection and evaluated stability against the digestive processes along with its uptake and transepithelial transport by intestinal epithelial cells. Milk exosomal siRNA were found resistant to different digestive juices, including saliva, gastric, bile, and pancreatic juices, in vitro and were internalized by Caco-2 cells. The stable delivery of exosomal AF-488 siRNA along with its transepithelial transport was confirmed by fluorescence microscopy and fluorescence intensity measurements. In summary, the encapsulation of siRNA in milk exosomes resists harsh digestive processes, improving intestinal permeability and payload protection.

Direct saliva transcript analysis as a novel non-invasive method for oestrus marker detection in buffaloes.

Abstract Salivary RNA-based biomarkers are not available for any physiological condition in farm animals. Hence, an objective of this study was to perform salivary transcript analysis in buffaloes. Saliva, after removal of the cells and particulate matter, was directly used for RT-PCR without RNA isolation. Direct saliva transcript analysis (DSTA) showed a suggestively significant higher expression of the Heat shock protein 70 (HSP70) and Toll-like receptor 4 (TLR4) at oestrus than the diestrous period in buffaloes by a non-parametric Mann-Whitney U test. Therefore, DSTA without RNA isolation is an easy method to identify salivary RNA markers for oestrus detection in buffaloes.

A highly efficient method for extracting next-generation sequencing quality RNA from adipose tissue of recalcitrant animal species

The next‐generation sequencing (NGS) based RNA sequencing (RNA‐Seq) and transcriptome profiling offers an opportunity to unveil complex biological processes. Successful RNA‐Seq and transcriptome profiling requires a large amount of high‐quality RNA. However, NGS‐quality RNA isolation is extremely difficult from recalcitrant adipose tissue (AT) with high lipid content and low cell numbers. Further, the amount and biochemical composition of AT lipid varies depending upon the animal species which can pose different degree of resistance to RNA extraction. Currently available approaches may work effectively in one species but can be almost unproductive in another species. Herein, we report a two step protocol for the extraction of NGS quality RNA from AT across a broad range of animal species.

Three‐dimensional culture of buffalo granulosa cells in hanging drop mimics the preovulatory follicle stage

Granulosa cell (GC) culture models mimicking the intrafollicular environment are limited. Such models have a great potential in reproductive toxicity studies. The buffalo, a monovulatory species like humans, could be a better model than polyovulatory rodents. Therefore, we targeted the development and characterization of three‐dimensional (3D) culture systems for buffalo GCs. The GCs from small ovarian follicles (SF) maintained the CYP19 gene expression for 144 hr in a 2D culture system. Hence, GCs from SF were cultured directly in 3D using hanging drop and Poly‐([2‐hydroxyethyl methacrylate]) (polyHEMA) methods in the DMEM media containing 1 ng/ml FSH and 10 ng/ml IGF‐1 for 144 hr. The expression profile of nine GC‐specific transcripts; CYP19, TNFAIP6, AMH, PTI, NR4A1, FSHR, RUNX, LHR, and COX2/PTGS2; revealed that 3D‐spheroids developed in hanging drop method maintained the GC phenotype of preovulatory follicles. Therefore, hanging drop method is a best method for culturing GCs to mimic the intrafollicular environment.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP), a light for mammalian transcript analysis in low-input laboratories

Transcript analysis is usually performed by costly, time‐consuming, and expertise intensive methods, like real time‐PCR, microarray, etc. However, they are not much feasible in low‐input laboratories. Therefore, we implemented the reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) as a means of mammalian transcript analysis. Particularly, RT‐LAMP was developed for buffalo aromatase cytochrome P450 (CYP19) transcript, to study its expression in 3D‐cultured buffalo granulosa cells, which were exposed to lipopolysaccharide (LPS). The CYP19‐RT‐LAMP assay rapidly identified the LPS‐induced downregulation of the CYP19 gene within 30 min at 63°C in a water bath. The assay was visualized via unaided eye by observing the change in turbidity and fluorescence, which were decreased by increasing the LPS exposure time to granulosa cells. Overall, the developed CYP19‐RT‐LAMP assay provided a hope on the application of RT‐LAMP for mammalian transcript analysis in low‐input laboratories.

Differentially expressed miRNA-210 during follicular-luteal transition regulates pre-ovulatory granulosa cell function targeting HRas and EFNA3†

Ovarian folliculogenesis, ovulation, and luteinization are an important prerequisite for fertility performance in mammals. Spatial and temporal key factors and proteins for their regulation are well known. Recent advancement in the field of molecular biology led to the discovery of another class of gene regulators, microRNA (miRNA). Previous studies on profiling of miRNA in buffalo ovaries revealed that miRNA‐210 (miR‐210) is differently expressed in follicular‐luteal transition. Therefore, the present study was planned to ascertain the role of miR‐210 in buffalo granulosa cells. Cultured granulosa cells were transfected with miR‐210 mimic. Effect of overexpression of miR‐210 was analyzed on granulosa cell marker genes (CYP19A1 and PCNA) which were significantly downregulated (P < 0.05). Further, target genes of miR‐210 were screened using Target Scan software v7.1 and a list of 37 genes with cumulative weight context score (CWCS) > 0.5 was sorted followed by their functional annotation and network analyses using PANTHER and STRING software. Bioinformatics analyses identified HRas gene as a potential hub gene of miR‐210 targeted genes. HRas has been shown to be involved in diverse biological pathways regulating ovarian functions. An expression analysis of HRas was further validated both in vitro and in vivo. EFNA3 (EFHRIN‐A3), another identified target of miR‐210 known to be involved in angiogenesis, was also downregulated in miR‐210 transfected granulosa cells. In conclusion, the present study demonstrated that miR‐210 can regulate granulosa cell function at preovulatory stage through HRas and EFNA3. Further studies are needed to find the mechanism how miR‐210 regulates the granulosa cells function through these targets.