Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

Design Maps of TRPM8 as a Thermometer for Cold Detection

The menthol sensor TRPM8 can be activated by cold and thus serves as a thermometer in a primary afferent sensory neuron for noxious cold detection. However, the underlying design principle is unknown. Here, a hairpin topological structural model and graph theory were prepared to test a role of the cold-dependent hairpin formation in the cold-evoked gating pathway of TRPM8. The results showed that the formation of a large lipid-dependent hairpin initiates a low temperature threshold in favor of TRPM8 activation. Furthermore, two smaller hairpins, which enhance the coupled interactions of the voltage-sensor-like domain with both the pore domain and the TRP domain, can stabilize the cold efficacy and work as a fuse to prevent cold denaturation. The cold-induced hairpin rearrangements along the gating pathway may be necessary for the high cold sensitivity. This hairpin model may provide a structural basis for activation of the thermo-gated TRP channels at low temperature.

Identification of Genes Related to Cold Tolerance and Novel Genetic Markers for Molecular Breeding in Taiwan Tilapia (Oreochromis spp.) via Transcriptome Analysis

Taiwan tilapia is one of the primary species used in aquaculture practices in Taiwan. However, as a tropical fish, it is sensitive to cold temperatures that can lead to high mortality rates during winter months. Genetic and broodstock management strategies using marker-assisted selection and breeding are the best tools currently available to improve seed varieties for tilapia species. The purpose of this study was to develop molecular markers for cold stress-related genes using digital gene expression analysis of next-generation transcriptome sequencing in Taiwan tilapia (Oreochromis spp.). We constructed and sequenced cDNA libraries from the brain, gill, liver, and muscle tissues of cold-tolerance (CT) and cold-sensitivity (CS) strains. Approximately 35,214,833,100 nucleotides of raw sequencing reads were generated, and these were assembled into 128,147 unigenes possessing a total length of 185,382,926 bp and an average length of 1446 bp. A total of 25,844 unigenes were annotated using five protein databases and Venny analysis, and 38,377 simple sequence repeats (SSRs) and 65,527 single nucleotide polymorphisms (SNPs) were identified. Furthermore, from the 38-cold tolerance-related genes that were identified using differential gene expression analysis in the four tissues, 13 microsatellites and 37 single nucleotide polymorphism markers were identified. The results of the genotype analysis revealed that the selected markers could be used for population genetics. In addition to the diversity assessment, one of the SNP markers was determined to be significantly related to cold-tolerance traits and could be used as a molecular marker to assist in the selection and verification of cold-tolerant populations. The specific genetic markers explored in this study can be used for the identification of genetic polymorphisms and cold tolerance traits in Taiwan tilapia, and they can also be used to further explore the physiological and biochemical molecular regulation pathways of fish that are involved in their tolerance to environmental temperature stress.

Tolerable range of abdomen and waist skin temperature for heating-capable smart garments

Purpose This study aims to provide information on how to monitor the temperature setting of a heating device in order to implement a heating unit successfully in the smart clothing by observing voluntary heating behavior of wearers.Design/methodology/approach Subjects wearing base layers and additional clothing were asked to turn on and off the switch when wanted in the cold environmental chamber. Tolerable range of skin temperature (TST) depending on the location of body was obtained by observing the temperature at the time when the heating device was turned on and off during a rest–running–rest protocol.Findings The TST was 32.8–49.4 °C and decreased to 31.3–37.6 °C around abdomen and back waist, respectively. Changes in the wearers' voluntary control behavior were observed depending on the individual's level of cold-sensitivity and activity level of rest and running. TST was 35.8–49.4 °C (Rest 1: rest before exercise), 40.0–42.0 °C (Running) and 35.3–43.2 °C (Rest 2: rest after exercise) for cold-sensitive group, whereas it was 32.8–36.2 °C (Running) and 34.4–45.7 °C (Rest 2: rest after exercise) for cold-insensitive group.Originality/value In this study, results with detailed body locations and wearer's thermal sensitivity provide practical references for the implementation of a heating device to the comfortable multilayered smart clothing.

Differential Cutaneous Thermal Sensitivity in Humans: Method of Limit vs. Method of Sensation Magnitude

Introduction: The method of limits (MLI) and method of level (MLE) are commonly employed for the quantitative assessment of cutaneous thermal sensitivity. Thermal sensation and thermal comfort are closely related and thermal sensations evoked from the peripheral thermoreceptors play an important role in thermoregulatory response to maintain normal body temperature. The purpose of this study was to compare the regional distribution of cutaneous warm and cold sensitivity between MLI and the method of sensation magnitude (MSM). Method: Twenty healthy men completed MLI and MSM to compare the regional distribution of cutaneous warm and cold sensitivity in the thermal neutral condition. The subjects rested on a bed in a supine position for 20 min. Next, the cutaneous thermal sensitivity of ten body sites was assessed by the means of MLI and MSM for both warmth and cold stimuli. Results: The absolute mean heat flux in MLI and thermal sensation magnitude in MSM showed significantly greater sensitivity to cold than to warm stimulation (p < 0.01), together with a similar pattern of regional differences across ten body sites. Both sensory modalities indicated acceptable reliability (SRD%: 6.29–8.66) and excellent reproducibility (ICC: 0.826–0.906; p < 0.01). However, the Z-sore distribution in MSM was much narrower than in MLI, which may limit the test sensitivity for the detection of sensory disorders and/or comparison between individuals. Conclusion: The present results showed that both MLI and MSM are effective means for evaluating regional cutaneous thermal sensitivity to innocuous warm and cold stimulations to a strong degree of reliability and reproducibility.

Manual Therapy With Rest as a Treatment for Established Inflammation and Fibrosis in a Rat Model of Repetitive Strain Injury

Background: Repetitive strain injuries caused by repetitive occupational work are difficult to prevent for multiple reasons. Therefore, we examined the effectiveness of manual therapy (MT) with rest to treat the inflammation and fibrosis that develops through the performance of a repetitive task. We hypothesized that this treatment would reduce task-induced sensorimotor declines and neuromuscular inflammation.Methods: Twenty-nine female Sprague-Dawley rats performed a reaching and lever-pulling task for 14weeks. All ceased performing the task at 14weeks. Ten were euthanized at this timepoint (TASK). Nine received manual therapy to their upper extremities while resting 7weeks (MTR); 10 were assigned to rest alone (REST). Ten additional food restricted rats were included that neither performed the task nor received manual therapy (FRC).Results: Confirming previous experiments, TASK rats showed behavioral changes (forepaw mechanical hypersensitivity, reduced grip strength, lowered forelimb/forepaw agility, and noxious cold temperature sensitivity), reduced median nerve conduction velocity (NCV), and pathological tissue changes (myelin degradation, increased median nerve and muscle inflammation, and collagen production). Manual therapy with rest (MTR) ameliorated cold sensitivity seen in REST rats, enhanced muscle interleukin 10 (IL-10) more than in REST rats, lead to improvement in most other measures, compared to TASK rats. REST rats showed improved grip strength, lowered nerve inflammation and degraded myelin, and lowered muscle tumor necrosis factor alpha (TNFα) and collagen I levels, compared to TASK rats, yet maintained lowered forelimb/forepaw agility and NCV, and increased neural fibrosis.Conclusion: In our model of repetitive motion disorder, manual therapy during rest had modest effects on behavioral, histological, and physiological measures, compared to rest alone. These findings stand in contrast to the robust preventive effects of manual therapy in this same model.

Outcomes of Surgery for Enchondromas within the Hand

Background. Enchondromas are the most common benign bone tumours found in the hand. They are usually accidentally diagnosed on an X-ray, because they grow asymptomatically. In some cases, a pathological fracture of the involved phalanx may be the first sign. The objective of this study was to assess the results of operative treatment of enchondromas involving hand phalanges and metacarpals. Material and methods. The study group consisted of 24 patients, 16 women (67%) and 8 men, (33%), aged a mean of 31 years, who were operated on at our centre. The surgery consisted in curettage, and - in most cases - filling the bone defect with either a bone graft or a bone substitute. Follow-up assessment was performed over the telephone in 17 patients (79% of the group) at a mean of 2 years after surgery. Results. Half of the patients were asymptomatic and fully recovered functionally, whereas the other half complained of some not troublesome symptoms such as scar discomfort, limitation of finger movement or cold sensitivity. No differences were observed with regard to the material used for filling of the bone defect following curettage. Two cases of recurrence were noted after surgery: one in the bone substitute group and one in the bone graft group. Conclusions. 1. Enchondromas are the most common benign bone tumours encountered in bones of the hand. 2. The first line treatment in these lesions is curettage and filling of the bone defect with a bone sub­stitute or cancellous bone graft. 3. Both the results of the present study and literature data show that the approach to managing the tu­mour cavity after curettage has no significant effect on outcomes, which are essentially satisfactory.

First report of Apple rubodvirus 2 infecting pear (Pyrus communis) in South Africa

Apple rubbery wood virus 2 (ARWV-2; Rott et al., 2018) belong to the species Apple rubodvirus 2, a member of the genus Rubodvirus (family Phenuiviridae; Kuhn et al 2020). ARWV-2 was first identified in apples and is associated with apple rubbery wood disease (ARWD) that is characterized by unusual flexibility of stems and branches, reduced growth, shortened internodes and increased cold sensitivity (Jakovljevic et al., 2017, Rott et al., 2018). ARWD was first reported in 1935 in England on apple and has since been found on quince and pear (Jakovljevic et al., 2017; Rott et al., 2018). In January 2021, leaves were collected from a pear tree (Pyrus communis cv. Forelle, F514) in a commercial orchard near Villiersdorp, South Africa. The tree displayed no foliar or tree branch symptoms, except for malformed fruits potentially due to insect feeding damage or pear stony pit disease previously associated with infection of apple stem pitting virus (ASPV) (Paunovic et al. 1999). Leaf petioles (one gram) were used for total RNA extraction, using a modified CTAB extraction protocol (Ruiz-García et al. 2019). A sequencing library was constructed (Illumina TruSeq Stranded Total RNA with plant Ribo-Zero) and sequenced on an Illumina HiseqX instrument (Macrogen, South Korea). A total of 30,709,182 paired-end reads (100 nt) were obtained and trimmed for quality with Trimmomatic (SLIDINGWINDOW:3:20, MINLEN:20) (Bolger et al. 2014). De novo assembly, using default parameters of CLC Genomics Workbench 11.0.1 (Qiagen), resulted in 97,294 contigs. BLASTn analysis identified 17 viral contigs, with 14 contigs having high nucleotide identity to ASPV and three to ARWV-2. The latter contigs included all three segments of ARWV-2. The L contig was 7371 nts, M was 1289 nts and S was 1463 nts in length, generated with 7341, 626 and 9161 reads for segment L, M and S, respectively. Segment S had the highest read coverage (524.87x), followed by segment L (88.07x) and M (36.60x). The ARWV-2 GenBank accessions with the highest percentage identity to the contigs were MF062128.1 from United States of America (98.2% to segment L), MN163134.1 from China (97.5% to segment M) and NC_055535.1 from Germany (93.5% to segment S). The contigs spanned 100%, 80.92% and 100% of these accessions of segments L, M and S, respectively and were deposited in GenBank as accessions MZ593725- MZ593727. Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the presence of ARWV-2 in sample F514, using primers directed at segments L (con708_178F/con708_666R), M (ARWaV-2S1_38F/ARWaV-2S1_682R) and S (ARWaV-2M567F/ARWaV-2M1342R) (Rott et al., 2018). Amplicon sequences (510 bp (L), 645 bp (M) and 799 bp (S)) were confirmed with bi-directional Sanger sequencing. Fifty-nine additional pear samples were surveyed in 2021 for ARWV-2 using the M segment assay as mentioned above. The survey included the Koue Bokkeveld and Elgin areas, and cultivars Bosc (22 samples), Abate (10 samples), Rosemarie (3 samples), Forelle (9 samples), Packham’s Triumph (12 samples) and Early Bon Chretien (3 samples). A total of 27 samples (11 samples from the Koue Bokkeveld region and 16 samples from the Elgin region) tested positive for ARWV-2, demonstrating the common presence of this virus in pears in South Africa. This is the first report of ARWV-2 infecting pear in South Africa. Although no association with disease symptoms were observed, this study expands the data on the incidence and distribution of this virus in South Africa.