Mycobacterium abscessus: insights from a bioinformatic perspective.

Mycobacterium abscessus is a nontuberculous mycobacterium, associated with broncho-pulmonary infections in individuals suffering from cystic fibrosis, bronchiectasis, and pulmonary diseases. The risk factors for transmission include biofilms, contaminated water resources, fomites, and infected individuals. M. abscessus is extensively resistant to antibiotics. To date, there is no vaccine and combination antibiotic therapy is followed. However, drug toxicities, low cure rates, and high cost of treatment make it imperfect. Over the last 20 years, bioinformatic studies on M. abscessus have advanced our understanding of the pathogen. This review integrates knowledge from the analysis of genomes, microbiomes, genomic variations, phylogeny, proteome, transcriptome, secretome, antibiotic resistance, and vaccine design to further our understanding. The utility of genome-based studies in comprehending disease progression, surveillance, tracing transmission routes, and epidemiological outbreaks on a global scale has been highlighted. Furthermore, this review underlined the importance of using computational methodologies for pinpointing factors responsible for pathogen survival and resistance. We reiterate the significance of interdisciplinary research to fight M. abscessus. In a nutshell, the outcome of computational studies can go a long way in creating novel therapeutic avenues to control M. abscessus mediated pulmonary infections.

Solid surface vitrification of goat testicular cell suspension enriched for spermatogonial stem cells.

This study reports solid surface vitrification (SSV) of goat testicular cell suspensions (TCS) enriched for spermatogonial stem cells (SSCs). The TCS was isolated from pre-pubertal goat testis by enzymatic digestion, enriched for SSCs by filtration and differential plating, and were vitrified-warmed by SSV. The study showed that SSV could successfully vitrify goat TCS although the percentage of live cells in the vitrified-warmed group was lower (74.8 ± 4.1%) than in non-vitrified control (80.6 ± 6.27%). The vitrified-warmed TCS formed putative SSC colonies upon their in vitro culture, but the colony size of vitrified-warmed cells (24.3 ± 1.8 µm) was smaller than those of non-vitrified warmed cells (58.4 ± 2.5 µm). Mitochondrial activity (0.40 vs. 0.38 A U.), population doubling time (33.45 ± 1.25 h vs. 31.86 ± 1.90 h), and the cell proliferation rate (0.72 ± 0.10 vs. 0.75 ± 0.11 per day) of total cells (including putative SSCs and other somatic cells) did not differ (p > 0.05) between control and SSV vitrified-warmed groups. However, during in vitro culture for 96 h, vitrified-warmed cells showed significantly lower (0.75 vs. 1.33 A U.; p < 0.05) mitochondrial activity than non-vitrified controls. The DCFDA assay showed that ROS activity was significantly (p < 0.05) higher in vitrified-warmed cells (52.8 ± 4.1 A U) than non-vitrified control cells (32.8 ± 2.1 AU). In conclusion, our results suggest that SSC-enriched goat TCS could be successfully cryopreserved by SSV. However, ROS-induced damages to cell cytoplasmic components reduce their cellular proliferation and require further improvement in the protocol. To the best of our knowledge, this study is the first report on the SSV of SSC-enriched goat TCS.

Strategies for cryopreservation of testicular cells and tissues in cancer and genetic diseases.

Cryopreservation of testicular cells and tissues is useful for the preservation and restoration of fertility in pre-pubertal males expecting gonadotoxic treatment for cancer and genetic diseases causing impaired spermatogenesis. A number of freezing and vitrification protocols have thus been tried and variable results have been reported in terms of cell viability spermatogenesis progression and the production of fertile spermatozoa. A few studies have also reported the production of live offspring from cryopreserved testicular stem cells and tissues in rodents but their replication in large animals and human have been lacking. Advancement in in vitro spermatogenesis system has improved the possibility of producing fertile spermatozoa from the cryopreserved testis and has reduced the dependency on transplantation. This review provides an update on various cryopreservation strategies for fertility preservation in males expecting gonadotoxic treatment. It also discusses various methods of assessing and ameliorating cryoinjuries. Newer developments on in vitro spermatogenesis and testicular tissue engineering for in vitro sperm production from cryopreserved SSCs and testicular tissue are also discussed.

Comparison of two culture methods during in vitro spermatogenesis of vitrified-warmed testis tissue: Organ culture vs. hanging drop culture.

Solid surface vitrification (SSV) is a cost effective and simple method for testis tissue preservation. Vitrified-warmed testis tissue was successfully cultured using various organ culture methods. In this study, we compared two culture methods viz. hanging drop (HD) and organ culture (OC) methods for in vitro spermatogenesis of goat testis tissue vitrified-warmed by SSV. It was observed that OC method was superior (p < 0.05) to HD method in terms of post-warming metabolic activity of testicular tissue, as measured by MTT assay on Day 7 and Day 14 of culture, respectively. The size of the tissue also played an important role in post-warming metabolic activity and viability (4 mm3: 72.7 ± 1.2% vs. 9 mm3: 62.7 ± 1.3% vs. 16 mm3: 40.5 ± 1.7%) of vitrified tissues with smaller tissue resulting in better result. The vitrification-induced ROS activity significantly decreased during their in vitro culture. Histology and scanning electron microscopy (SEM) showed the rupture of basal membrane, surface morphology and, cell loss due to vitrification. However, histology and immunohistochemistry showed the progression of in vitro spermatogenesis and formation of elongated spermatozoa in both fresh and vitrified-warmed testis tissue cultured by OC method. Taken together, our results suggest that OC method is superior to HD method for culturing goat testis tissue vitrified-warmed by SSV.

Cryopreservation of murine testicular Leydig cells by modified solid surface vitrification with supplementation of antioxidants.

Reports on the vitrification of somatic cells are scarce. Here, we show that Leydig cells (murine cell line TM3) could be successfully vitrified by both open vitrification [plastic straw (PS) and plastic vials (PV)] and closed ultravitrification [microdrop (MD) and solid surface vitrification (SSV)], after protocol optimization. However, open ultravitrification resulted in better post-warming viability than closed systems of vitrification with highest success obtained in modified SSV (84.8 ±â€¯1.86%; p < 0.05). Leydig cells vitrified-warmed by modified SSV also showed superior (p < 0.05) cell growth, mitochondrial activity and cytoplasmic esterase enzyme activity, than MD, PS and PV, respectively. It was also observed that vitrified-warmed cells had higher level of ROS activity than non-vitrified control cells (41.6 ±â€¯4.0 vs. 16.7 ±â€¯1.0; p < 0.05). Treatment of cells with glutathione (GSH) or 2-mercaptoethanol (2-ME) (0, 10, 50, 100 µM) significantly (p < 0.05) reduced the ROS activity but had no significant (p > 0.05) effect on post-warm viability. Nevertheless, antioxidant-treated cells had improved mitochondrial activity, cytoplasmic esterase activity and cell growth during in vitro culture (p < 0.05). In conclusion, our results suggest that modified SSV offers a viable method for vitrifying single cell suspension of Leydig cells. To the best of our knowledge, this is the first report on cryopreservation of Leydig cells by vitrification.

Evaluation of sodium alginate for encapsulation-vitrification of testicular Leydig cells.

This study reports encapsulation-vitrification of Leydig cells. The Leydig cells were encapsulated in sodium alginate beads of different sizes and cryopreserved by vitrification or slow freezing. Physico-chemical characterization of beads was done by Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), Fluorescence Recovery after Photobleaching (FRAP) and in vitro biodegradation study. Surface morphology of cryopreserved cell-encapsulated beads was evaluated by Environmental Scanning Electron Microscopy (E-SEM), encapsulation efficiency and viability of cells were assessed by Trypan blue assay, mitochondrial activity (MTT assay) and cytoplasmic esterase enzyme activity (FDA assay), respectively. Results showed that vitrification gives better results than slow freezing with respect to surface morphology as well as cell viability of the cell-encapsulated beads (86.94 ± 2.20% vs. 67.94 ± 2.30%; p < 0.05). Encapsulation of cells in small diameter beads (1.8 mm) gave a better cell proliferation rate than large (2.1 mm and 2.7 mm). There was a significant difference in the population doubling time (47.9 ± 1.7 h vs. 67.1 ± 2.5 h) and cell proliferation rate (0.50 ± 0.24 vs. 0.36 ± 0.24 per day) of vitrified-warmed cell encapsulated beads with different diameter (p < 0.05). Encapsualtion in sodium alginate beads is a promising method for cryopreservation of Leydig cells by slow freezing as well as vitrification.