DNA Replication Challenges: Telomeres and R loops

Faithful DNA replication and repair are essential for maintaining genome stability and preventing various diseases including cancer. Both processes are executed by numerous redundant mechanisms to ensure that these processes are uninterrupted even when a primary mechanism fails. Despite this, they are not immune to challenges and failures leading to DNA damage and genome instability. These problems are more evident at the difficult-to-replicate regions of the genome such as the telomeres that cap and protect linear chromosome ends. Additionally, topological structures such as RNA:DNA hybrids, commonly referred to as R loops, can also present severe challenges to the DNA replication and repair machineries. Herein we report the functions of two distinct DNA replication and repair proteins—flap endonuclease 1 (FEN1) and ribonuclease H1 (RNH1)—that preserve genome stability. First, we show that FEN1 limits telomere fragility in leading strand replicated telomeres. This is mediated by its flap endonuclease activity independent of its gap endonuclease activity and C-terminal interactions. We show that the fragility phenotype is increased by RNA polymerase II inhibition and rescued by ectopic RNH1 expression. Because the telomere is transcribed and can form hybrids, these data suggest that the FEN1-mediated telomere fragility depends on RNA:DNA hybrids that accumulate from co-directional replisome-RNAP collision at the leading strand replicated telomere. These findings are the first to assign a leading strand specific function of FEN1, which is a canonical lagging strand protein. Second, we uncover a novel role for human RNH1 in DNA replication in the nucleus. We show that RNH1 depletion results in a global DNA damage response as well as telomere loss phenotype. Because RNH1 resolves RNA:DNA hybrids, we measured those hybrid levels and found that they increase upon RNH1 depletion. Given these hybrids could pose barriers to a moving replication machinery, we interrogated replication efficiency and discovered that RNH1 facilitates the replication fork movement, possibly by clearing hybrids. These data shed light onto the role of RNH1 in global DNA replication. Together, our work underscore the complexity of DNA replication and repair processes and highlight the varied roles that FEN1 and RNH1 play to maintain genome stability

Ecotourism, indigenous people and their local environment: A case study about Amaltari community homestay Nawalparasi, Nepal

This case study deals with ecotourism and its impacts on the indigenous people of Amaltari,  Nawalparsi district of Nepal. Homestay is practiced by locals of different villages in Nepal. Amaltari is one of such villages which are home to the 'tharu' local group. This research work will unleash impacts on the local indigenous people. The research work deals with both primary and secondary data to find the realities. Several hosts of homestay ecotourism have been interviewed to find the real impacts of ecotourism on indigenous people. The finding has been supported by previous literature work which deals with similar topics. With the help of secondary data, it has been proved that the positive impacts are far more than the negative impacts of homestay ecotourism on the indigenous people of Amaltari, Nepal. The Finding s of the study showed that, socioeconomic status of people has improved, and they have found outof-box ways to earn a livelihood. In addition, socially, they have seen positive changes in themselves. Learning English is one of the major positives. Society has become inclusive, and homestay eco-tourism has helped women's empowerment and increased the prosperity of women. Research also finds that there are some threads to the culture of the locals; western culture is penetrating into the region and impacting young people.  Furthermore, there are some problems that need to be solved to make ecotourism more adaptable, including the relationship between society, the environment, and tourism.

Ecotourism, indigenous people and their local environment: A case study about Amaltari community homestay Nawalparasi, Nepal

This case study deals with ecotourism and its impacts on the indigenous people of Amaltari,  Nawalparsi district of Nepal. Homestay is practiced by locals of different villages in Nepal. Amaltari is one of such villages which are home to the 'tharu' local group. This research work will unleash impacts on the local indigenous people. The research work deals with both primary and secondary data to find the realities. Several hosts of homestay ecotourism have been interviewed to find the real impacts of ecotourism on indigenous people. The finding has been supported by previous literature work which deals with similar topics. With the help of secondary data, it has been proved that the positive impacts are far more than the negative impacts of homestay ecotourism on the indigenous people of Amaltari, Nepal. The Finding s of the study showed that, socioeconomic status of people has improved, and they have found outof-box ways to earn a livelihood. In addition, socially, they have seen positive changes in themselves. Learning English is one of the major positives. Society has become inclusive, and homestay eco-tourism has helped women's empowerment and increased the prosperity of women. Research also finds that there are some threads to the culture of the locals; western culture is penetrating into the region and impacting young people.  Furthermore, there are some problems that need to be solved to make ecotourism more adaptable, including the relationship between society, the environment, and tourism.

Regulation of Guinea Pig Detrusor Smooth Muscle Excitability by 17β-Estradiol: The Role of the Large Conductance Voltage- and Ca2+-Activated K+ Channels.

Estrogen replacement therapies have been suggested to be beneficial in alleviating symptoms of overactive bladder. However, the precise regulatory mechanisms of estrogen in urinary bladder smooth muscle (UBSM) at the cellular level remain unknown. Large conductance voltage- and Ca2+-activated K+ (BK) channels, which are key regulators of UBSM function, are suggested to be non-genomic targets of estrogens. This study provides an electrophysiological investigation into the role of UBSM BK channels as direct targets for 17β-estradiol, the principle estrogen in human circulation. Single BK channel recordings on inside-out excised membrane patches and perforated whole cell patch-clamp were applied in combination with the BK channel selective inhibitor paxilline to elucidate the mechanism of regulation of BK channel activity by 17β-estradiol in freshly-isolated guinea pig UBSM cells. 17β-Estradiol (100 nM) significantly increased the amplitude of depolarization-induced whole cell steady-state BK currents and the frequency of spontaneous transient BK currents in freshly-isolated UBSM cells. The increase in whole cell BK currents by 17β-estradiol was eliminated upon blocking BK channels with paxilline. 17β-Estradiol (100 nM) significantly increased (~3-fold) the single BK channel open probability, indicating direct 17β-estradiol-BK channel interactions. 17β-Estradiol (100 nM) caused a significant hyperpolarization of the membrane potential of UBSM cells, and this hyperpolarization was reversed by blocking the BK channels with paxilline. 17β-Estradiol (100 nM) had no effects on L-type voltage-gated Ca2+ channel currents recorded under perforated patch-clamp conditions. This study reveals a new regulatory mechanism in the urinary bladder whereby BK channels are directly activated by 17β-estradiol to reduce UBSM cell excitability

LC-HRMS-Based Profiling: Antibacterial and Lipase Inhibitory Activities of Some Medicinal Plants for the Remedy of Obesity

Globally, obesity is a serious health concern that causes numerous diseases, including type 2 diabetes, hypertension, cardiovascular diseases, etc. Medicinal plants have been used to aid in weight loss since ancient times. Thus, this research is focused on the exploration of pancreatic lipase inhibitory activity and secondary metabolite profiling of Bergenia ciliata, Mimosa pudica, and Phyllanthus emblica, selected based on an ethnobotanical survey. The lipase inhibition was investigated using 4-nitrophenyl butyrate (p-NPB) as a substrate. To uncover further therapeutic potentials of these medicinal plants, antimicrobial activity and minimum inhibitory concentration (MIC) of the extracts were also determined. The ethyl acetate plant extracts showed higher antimicrobial activity against Staphylococcus aureus, Escherichia coli, Salmonella typhi, and Shigella sonnei. The MIC of ethyl acetate extracts of medicinal plants considered in this study ranges from 1.56 to 6.25 mg/mL. The hexane fraction of Mimosa pudica and Phyllanthus emblica showed a higher lipase inhibitory activity as compared to others, with IC50 values of 0.49 ± 0.02 and 2.45 ± 0.003 mg/mL, respectively. In the case of Bergenia ciliata, the methanolic extract inhibited lipase more effectively than others, with an IC50 value of 1.55 ± 0.02 mg/mL (IC50 value of orlistat was 179.70 ± 3.60 µg/mL). A mass spectrometry analysis of various solvent/solvent partition fractions (extracts) revealed 29 major secondary metabolites. The research offers a multitude of evidence for using medicinal plants as antiobesity and antimicrobial agents

17β-Estradiol increases the frequency of TBKCs in freshly-isolated UBSM cells.

<p><b>A)</b> An original representative recording illustrating the stimulatory effects of 17β-estradiol (100 nM) on TBKC activity in an isolated UBSM cell. <b>B)</b> Summarized data illustrating the increase in TBKC frequency by 17β-estradiol (100 nM) in UBSM cells (n = 9, N = 9; *P<0.05).</p

17β-Estradiol increases depolarization-induced whole cell steady-state BK currents in freshly-isolated UBSM cells.

<p><b>A)</b> Representative traces from a voltage-clamp experiment illustrating that 17β-estradiol (100 nM) increases whole cell steady-state BK currents in an isolated UBSM cell. <b>B)</b> The current-voltage relationship curves illustrate the 17β-estradiol (100 nM)-mediated increase of whole cell steady-state BK currents in isolated UBSM cells (n = 11, N = 10; *P<0.05). <b>C)</b> Representative traces from a voltage-clamp experiment illustrating the lack of effects of 17β-estradiol (100 nM) on whole cell steady-state BK currents in the presence of the BK channel inhibitor paxilline (1 μM). <b>D)</b> The current-voltage relationship curves illustrate that 17β-estradiol (100 nM) had no significant effects on whole cell steady-state BK currents in the presence of 1 μM paxilline (n = 14, N = 12; P>0.05).</p