Madan, D. | Search bioRxiv

bioRxiv2025

One-Pot Isothermal Linear Amplification and Cas12a-based Nucleic Acid Detection

CRISPR-based nucleic acid diagnostics are a promising class of point-of-care tools that could dramatically improve healthcare outcomes for millions worldwide. However, these diagnostics require nucleic acid pre-amplification, an additional step that complicates deployment to low resource settings. Here, we developed CATNAP (Cas trans-nuclease detection of amplified products), a method that integrates isothermal linear DNA amplification with Cas12a detection in a single reaction. CATNAP uses a nicking enzyme and DNA polymerase to continuously generate single-stranded DNA, activating Cas12as trans-cleavage activity without damaging the template. We optimized enzyme combinations, buffer conditions, and target selection to achieve high catalytic efficiency. CATNAP successfully distinguished between high- and low-risk HPV strains and detects HPV-16 in a cervical cancer crude cell lysate at room temperature with minimal equipment, offering advantages over PCR-based approaches. We conclude that CATNAP bridges the sensitivity gap in CRISPR diagnostics while maintaining simplicity, making accurate disease detection more accessible in resource-limited settings.

biochemistry↗

bioRxiv2024

Homeostatic balance of Gut-resident Tregs (GTregs) plays a pivotal role in maintaining bone health under post-menopausal osteoporotic conditions

Osteoporosis is a skeletal condition characterized by the deterioration of bone tissue. The immune system plays a crucial role in maintaining bone homeostasis and combating the development of osteoporosis. Immunoporosis is the term used to describe the recent convergence of research on the immune systems role in osteoporosis. Gut harbors the largest component of the immune system and there is growing evidence that intestinal immunity plays a vital role in regulating bone health. Gut-resident regulatory T cells (GTregs) play an essential role in inhibiting immune responses and preventing various inflammatory manifestations. Our findings show that GTregs have a pivotal role in the pathophysiology of post-menopausal osteoporosis (PMO). We investigated the potential of GTregs in regulating the development of bone cells in vitro. We observed that GTregs significantly enhance osteoblastogenesis with concomitant inhibition of osteoclastogenesis in a cell-ratio-dependent manner. We further report that the deficiency of short-chain fatty acids (SCFAs) in osteoporotic conditions substantially disrupts the composition of GTregs, leading to a loss of peripherally derived Tregs (pTregs) and an expansion of thymus-derived Tregs (tTregs). Moreover, the administration of probiotics Lactobacillus rhamnosus and Bifidobacterium longum modulated the GTregs compartment in an SCFA-dependent manner to mitigate inflammatory bone loss in PMO. Notably, SCFAs-primed GTregs were found to be significantly more effective in inhibiting osteoclastogenesis compared to unprimed GTregs. Altogether our results, for the first time, highlight the crucial role of GTregs in the pathophysiology of PMO, with potential clinical implications in the near future. O_FIG O_LINKSMALLFIG WIDTH=145 HEIGHT=200 SRC="FIGDIR/small/612848v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@b36b96org.highwire.dtl.DTLVardef@15c7ea8org.highwire.dtl.DTLVardef@17f5186org.highwire.dtl.DTLVardef@1c1438b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO A) During normal physiological conditions there is a sufficient proportion of Tregs in the mice intestine with a higher frequency of pTregs compared to the tTregs. Gut-resident Tregs inhibit osteoclastogenesis and therefore prevent bone loss. However post-menopausal condition decreases the Treg population in the intestinal tissues and further perturbs the balance of pTregs and tTregs resulting in bone loss. Probiotics such as Lactobacillus rhamnosus (LR) and Bifidobacterium longum (BL) induce the development of Tregs from naive T cells and further restore the balance of pTregs and tTregs in a short chain fatty acid (SCFA) dependent manner. SCFAs primed Tregs are more potent in inhibiting osteoclastogenesis thereby preventing bone loss due to osteoporosis. B) Similar to mice the frequency of Tregs decreases in the human colon of inflammatory bowel disease (IBD) patients with osteopenia or osteoporosis compared to the IBD patients without osteopenia/osteoporosis C_FIG

immunology↗