Purkayastha, D. | bioRxiv Science

bioRxiv2025

The regulation of virulence gene expression is controlled by phase separation of heterochromatin protein 1 (HP1) in Plasmodium falciparum

Malaria caused by Plasmodium falciparum involves antigenic variation on the infected red blood cell surface by a mutually exclusive expression of virulence (var) genes. The repressed var genes have a distinct genome organization where they localize as a cluster near the nuclear periphery and are bound by PfHP1 (P. falciparum Heterochromatin Protein 1). However, the mode of regulation of var genes by PfHP1 remains unclear. In this study, we show that PfHP1 undergoes phase separation in vitro in an RNA and DNA-dependent manner. Single-molecule DNA tethering experiments further revealed that AT-rich DNA sequences act as nucleation sites for the assembly and compaction of PfHP1-mediated heterochromatinization. We have also identified point mutations in the IDRs (intrinsically disordered regions) of PfHP1 that disrupt its phase separation as well as DNA compaction in vitro. To assess the dynamic properties of PfHP1 condensates in vivo, we performed fluorescence recovery after photobleaching (FRAP) in GFP tagged PfHP1 parasites, which revealed rapid fluorescence recovery, supporting their fluidity and phase separation behaviour. Ectopic expression of PfHP1 phase separation and DNA compaction mutants led to dispersed nuclear localization of PfHP1, in contrast to the punctate appearance of the wild-type protein and altered chromatin binding at var genes. These results were corroborated with DiCre/loxP-based conditional expression of the same PfHP1 mutants, which also led to the de-repression of multiple var genes (as many as 54 out of 60 var genes), mimicking the phenotype of PfHP1 depletion mutants. Hi-C sequencing of PfHP1 mutants revealed loss of interactions in the heterochromatic regions, indicating that PfHP1 phase separation is an essential mechanism for repressive cluster formation. In conclusion, our study demonstrates the role of PfHP1-mediated phase separation in heterochromatin formation and var gene silencing; unveiling a fundamental mechanism that drives antigenic variation in P. falciparum.

molecular biology↗

bioRxiv2022

Hand cold pressor test induces thermogenesis in upper thoracic regions as measured by skin surface infrared thermography

BackgroundCold exposure may cause health problems and impaired productivity in outdoor or cold-temperature workers. The cold pressor test (CPT) is a laboratory procedure that measures cardiovascular and thermoregulatory responses to acute cold exposure such as metabolic activity in brown adipose tissue. How the body responds to acute cold exposure of a hand is not completely understood. We tested the hypothesis that the upper thorax produces heat during a single hand-CPT, which restores warmth to the cold-expose appendage. ObjectivesThe objective was to measure skin temperature changes in the upper thoracic regions and the cold-exposed appendage during a CPT. The secondary objective was to determine if cardiovascular or psychological responses during CPT accounted for skin temperature changes. Methods50 healthy participants immersed their right hand up to wrist level in 4 {degrees}C water for three minutes. Surface skin temperatures were imaged by infrared thermography at baseline, during CPT, and in the recovery phase. Sublingual oral temperature and water bath temperature were recorded throughout the test. Cardiovascular responses were monitored by continuous finger pulse-wave plethysmography. Peak pain and peak stress were reported by the participants on a Likert scale. ResultsCPT increased the systolic blood pressure (+22 mmHg, p < 0.001), diastolic blood pressure (+15 mmHg, p < 0.001) and heart rate (+7 beats per minute, p = 0.024). During CPT, skin temperature increased on thoracic regions including mediastinal (+0.5{degrees}C, p < 0.021), sternal (+0.5{degrees}C, p < 0.002), right supraclavicular (+0.3{degrees}C, p < 0.042) and left supraclavicular (+0.3{degrees}C, p < 0.016) regions. During CPT, the hand was cooler on ventral (-14.6{degrees}C p < 0.001) and dorsal (-15.2{degrees}C p < 0.001) sides, and warmed up during recovery. The ventral forearm, dorsal forearm, antecubital fossa, and adjacent medial epicondyle region were significantly cooler throughout the recovery time. The oral temperature did not change during CPT. There were no correlations between the change in mediastinal skin temperature and the sex of the participant, or changes in cardiovascular parameters, peak pain, or peak stress values. ConclusionsLocalized hand cooling caused a rapid warming of the thorax, dissipation of cold in the forearm, and rewarming of the hand during recovery. Thermoregulation was not dependant on pain, stress, sex, or cardiovascular changes between participants. By understanding thermoregulation, better approaches can be developed to mitigate the negative impacts of cold exposure.

physiology↗