Cry Genes Research Articles

The genus Bacillus as biological control agent against pests and pathogens for sustainable agriculture

Background/Objective. Bacteria of the Bacillus genus have been studied since their discovery in 1872, for their ability to synthesize metabolites of interest such as proteins used to control phytopathogens. The objective of this research was to analyze the most outstanding species of the Bacillus genus, as well as the main compounds produced by these species, and the perspectives on the use of this bacterial genus for pest and disease control. Materials and Methods. An exhaustive search was carried out in scientific articles and books to gather the most relevant information regarding the Bacillus genus, focusing on its role as a biological control agent for pests and pathogens. Results. The Bacillus genus includes more than 427 taxa, which can be classified into different groups. Among the biological control agents (BCA) are the Bacillus cereus group, which includes B. cereus, B. anthracis, and B. thuringiensis, and the B. subtilis group, which includes B. subtilis, B. licheniformis, and B. pumilus, mainly. B. thuringiensis, through cry genes, has molecular mechanisms to synthesize a crystalline inclusion during sporulation, which contains proteins known as endotoxins or Cry proteins. B. subtilis produces substances with a high potential for biological control, such as volatile organic compounds, as well as bioactive secondary metabolites. Conclusion. The potential of the Bacillus genus to be used as biological control agents is evident. They are widely used for the development of different biopesticides that have advantages over other products. However, it is necessary to continue conducting research from the in vitro area in the laboratory to the field, to help guarantee their biosecurity and effectiveness.

Isolation and characterization of halophilic Bacillus thuringiensis from local Egyptian sites and their potential against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

BackgroundBacillus thuringiensis (Bt) serves as a peculiar soil microbe that is Gram-positive, rod-shaped, spore-forming, and aerobic facultative. It endures across a range of conditions and has been demonstrated to be an effective biological pest control agent against several insect pests. The main objective of the study is to isolate and recognize Bt bacteria from saline environments in Egyptian soil for biological surveillance uses.ResultsIsolates three, four, five, ten, eleven, twelve, and thirteen were found to be moderately tolerant, while isolate eight showed highly tolerant, but isolate six was found to be borderline extreme halophile. On the other hand, the other isolates showed extreme halophiles. To find out the presence of crystals, a scanning electron microscope was carried out and the result showed that the crystals ranged from spherical to bi-pyramidal spherical. Molecular analysis was carried out using universal primers to confirm the presence of cry genes. The results showed that 84.6% of the isolates contain cry seven and eight, whereas cry one and four are distributed by 92.3%. Cry two genes were found to be 100 percent in all tested and isolated cultures. Cry genes were present and distribution among isolated was detected using gene-specific primers. cry3Ba1 and cry8B showed the lowest and equal distribution among the isolates, while the cry2Ab2gene frequency distribution was (84.6%) among isolated cultures. Around six cry genes were found to be absent in all isolated cultures. The bioinsecticidal activity and bioassays were carried out to check the potential effect of halophilic Bt isolates against 1st instar larvae of the Fall Armyworm (FAW) Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). Mortality was calculated after infecting S. frugiperda with thirteen Bt isolates. The most virulent Bt isolate was isolate 7, followed by isolate 10, with LC50 values of 9.542 × 107 spores/ml and 1.289 × 108 spores/ml, respectively, after 72 h. Morphological traits of the larvae were carried out, to check the effect of Bt isolates as a biological control against the FAW.ConclusionTo protect and enhance the sustainability of field crops and vegetables, especially in new high salinity lands, from disease-causing insects such as FAW, it is necessary to identify new strains of Bt from these saline lands. In this field, local isolates of Bt from several regions in Egypt known for their high levels of salinity were documented and showed an effective bioinsecticide on FAW larvae.

Clinical Implication of Time of Ischaemic Stroke Among Post-Stroke Survivors from Eastern India: A Circadian Perspective.

The circadian variation in stroke occurrence is a well-documented phenomenon. However, the circadian effect on stroke outcome, particularly on post-stroke cognition, has not yet been fully elucidated. We aim to evaluate the influence of diurnal variation of stroke onset upon post-stroke cognition and development of post-stroke depression. Based on 4-hourly time period of stroke occurrence, 249 recruited cohorts were categorized into 6 groups. Several clinical and cognitive parameters were compared among the groups. Then, the mRNA expression of core clock genes in Peripheral Blood Mononuclear Cells were quantified and correlated with post-stroke outcomes among 24 acute phase cases with day-time or night-time stroke occurrence. Furthermore, the genetic susceptibility towards a higher number of cases in the morning was examined by genotyping CLOCK (rs1801260T/C, rs4580704G/C) and CRY2 (rs2292912C/G) genes variants in cases and 292 controls. In our study, the peak for highest incidence although observed during the early morning from 4 to 8 am, the nocturnal-onset stroke cases showed more severity (12.2 ± 5.67) at the time of admission irrespective of arterial territory involved. The night onset cases were also found to be more susceptible to develop language impairment and post-stroke depression in due course of time. Upon transcript analysis, circadian genes (BMAL1 and CRY1) were found to be downregulated in night-time cases than day-time ones during the acute phase of onset. In addition, those mRNA levels also showed a correlation with raw scores for language and depression. However, the difference in incidence frequency along a day did not reveal any genetic correlation. Therefore, we suggest night-time stroke to be positively associated with higher immediate severity and poor cognitive outcome than day-time injury and propose downregulation of circadian genes during the acute phase could be the underlying molecular mechanism for this.

Moderating effects of clock genes DNA methylation on the relationship between physical activity trajectories and depressive symptoms among Chinese college students.

To explore the 2-year physical activity trajectory of Chinese college students and further examine the longitudinal relationship between physical activity trajectory and depressive symptoms. Furthermore, our study aimed to clarify the potential role of clock genes deoxyribonucleic acid (DNA) methylation in the association between physical activity and depressive symptoms. From April 2019 to May 2021, College Student Behavior and Health Cohort Study was conducted among 1,179 students from two universities in Anhui and Jiangxi provinces. Latent Class Growth Model was applied to simulate and group physical activity in a total of five surveys. The PROCESS macro for SPSS was used to analyze the moderating effects of clock genes DNA methylation on the association between physical activity and depressive symptoms. Two physical activity trajectories were identified: "continued high level" and "gradual low level." Multinomial logistic regression analysis showed that the "gradual low level" of physical activity trajectory was independently associated with moderate-to-severe depressive symptoms. Furthermore, DNA methylation of the PER2 and CRY1 genes had negatively moderating effects between physical activity and depressive symptoms, and there was sex-specific effect for CRY1 gene. Our findings suggests that long-term low physical activity was connected to more severe depressive symptoms among college students, and clock genes DNA methylation may play a negative moderating role, attenuating the positive effect of high intensity physical activity levels on depressive symptoms. In this regard, intervention programs regarding depressive symptoms among Chinese college students should consider multiple approaches such as increasing the duration and intensity of physical activity. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Time-restricted feeding modulates gene expression related with rhythm and inflammation in Mongolian gerbils

Time-restricted feeding (TRF) has the potential to modulate circadian rhythm and widely studied in humans and laboratory mice. However, less is known about the physiological responses to TRF in wild mammals. Here, we used Mongolian gerbils, Meriones unguiculatus, to explore the effect of 6-week TRF on gene expression related with circadian rhythm and inflammation. The TRF gerbils had higher cumulative food intake than the ad libitum (AL) group, but body mass, feeding frequency/time and metabolic rate did not differ between groups. In the hypothalamus, downregulation of rhythm-related genes Per3, Cry1 and Dbp was detected in the daytime-restricted feeding (DRF) group and Cry1 was downregulated in the nighttime-restricted feeding (NRF) group. In the liver, the expression of Per1/3, Rev-erbα/β and Dbp was lower, and Bmal1 was higher in the DRF than in AL group, while NRF gerbils showed no changes. In the colon, the expression of Bmal1 and Cry1 was higher but Per3, Rev-erbα/β and Dbp were lower in the DRF than in AL group. Further, the expression of inflammation-related genes such as NF-κB, IL-1β, IL-18 and Nlrp3 was lower in the liver of DRF gerbils, and IL-1β was lower both in the hypothalamus and liver of NRF gerbils. Moreover, the genes related with inflammation such as NF-κB, Nlrp3, IL-10/18/1β and Tnf-α were positively or negatively correlated with multiple rhythm-related genes in the central and peripheral organs. In conclusion, TRF, particularly DRF, could modulate rhythm-related genes in the central and peripheral tissues and reduce hepatic expression of inflammation-related genes in gerbils.

Plasmid vector(s) in Bacillus thuringiensis harbor genes for insect pest control and for neglected infectious diseases in humans

Plasmids (circular DNA molecules) represent an ingenious strategy for horizontal gene transfer in prokaryotes and eukaryotic cells. Plasmids harbored in bacteria are responsible for the spread of traits such as antibiotic resistance, virulence factors, and the machinery for the horizontal gene transfer e.g., type IV secretion systems. Remarkably, Bacillus thuringiensis (Bt) cryptic plasmids encode and carry genes that, under the host environment, replicate and concomitate with sporulation, producing parasporal crystalline proteins of two major types, crystalline (Cry) and cytolytic (Cyt), the former toxic against different orders of insects such as Lepidopterans, Coleopterans, and Dipterans (Cry proteins, MW 50–130 KDa); Cyt proteins, produced by B. thuringiensis subspecies israelensis (Bti)(MW 27-kDa) are toxic against Dipterans, i.e., mosquitoes and black flies. The X-Ray tridimensional structure for both types of toxins, formed by three domains, mostly of beta sheets antiparallel (Domain II and Domain III) linked through loops of different lengths. Domain I is a bundle of alpha helices. This structure is characterized by five conserved blocks, implying a conservation in the mode of action. Cyt proteins possess two alpha helices and some beta sheets with a structure similar to the antimicrobial peptides. Indeed, the mode of action proposed is mediated by the toxin-lipid interaction that hypothetically could result in transmembrane ionic channel formation. Several pieces of evidence support the action of both toxins in insects and mammals. The question is to what extent these Bt/Bti plasmid-encoded Cry or Cyt genes can be applied as bioinsecticides individually or in combination with Lysinibacillus sphaericus. The feasibility of being considered a promising and safe biological strategy for crop pests and vector-borne neglected infectious diseases is an issue pinpointed in the present review.

In Vitro Evaluation of Native <i>Bacillus thuringiensis</i> (Berliner) Isolates against Different Insects

Evaluation of native strains of Bacillus thuringiensis (Berliner) isolates against different orders of insects and characterization of responsible Cry genes was carried out. Among the native isolates, BGC-1 showed the least LC50 value of 5.24 μg/ ml and was comparable to the reference strain HD1 (2.89 μg/ ml) against tobacco caterpillar Spodoptera litura (Fabricius). The isolate RCM-1 recorded least LC50 value of 4.69 μg/ ml against the housefly Musca domestica Linnaeus. One isolate viz., GHB-1 was found to be potential against both the larvae of S. litura and M. domestica. The isolate RCM-2 registered the least LC50 value of 8.21 μg/ ml against grubs of red flour beetle Tribolium castaneum (Herbst). Several isolates had more than one Cry gene in them. Among them, 12 isolates (63.15%) were found to contain Cry3 genes, 11 isolates harbour Cry4 genes, five isolates had Cry1, Cry2 and Cry11 genes each and four isolates contain Cry7 genes each. Native isolate, GHB-1 had Cry1, Cry3, Cry4 and Cry11 genes.

<i>Bacillus thuringiensis</i> Isolates and their Cry Genes Toxic to Chickpea Pod Borer <i>Helicoverpa armigera</i> (Hubner) from Ethiopia

Helicoverpa armigera (Hubn) is one of the most destructive insect pests of chickpea in Ethiopia. For sustainable management of insect pests of food crops, Bacillus thuringiensis (Bt) is a widely used bioinsecticide. This study was aimed at exploring indigenous Bt isolates that harbour cry genes to control H. armigera. Ten indigenous Bt isolates were analyzed for their cry genes. Accordingly, all the indigenous Bt isolates were observed to harbour two or more cry genes. Statistically significant (p<0.05) variations were observed among Bt species in influencing larval incidence, pod damage (%) and grain yield (t/ ha). Three potential indigenous Bt isolates were identified with their respective cry genes that included KDL (cry2 + cry4), AUGHS-1 (cry1 + cry4), and AUSD-1 (cry1 + cry2 + cry4 + cry7, 8 + cry9). Indigenous Bt isolates exhibited a strong potential in the management of chickpea pod borer. Development of commercial bioinsecticide and other Bt technologies using B. thuringiensis from Ethiopian sources will be a new avenue to be addressed.

Insights into the whole genome sequence of Bacillus thuringiensis NBAIR BtPl, a strain toxic to the melon fruit fly, Zeugodacus cucurbitae.

Bacillus thuringiensis is the most widely used biopesticide, targets a diversity of insect pests belonging to several orders. However, information regarding the B. thuringiensis strains and toxins targeting Zeugodacus cucurbitae is very limited. Therefore, in the present study, we isolated and identified five indigenous B. thuringiensisstrains toxic to larvae of Z. cucurbitae. However, of five strains NBAIR BtPl displayed the highest mortality (LC50 = 37.3μg/mL) than reference strain B. thuringiensis var. israelensis (4Q1) (LC50 = 45.41μg/mL). Therefore, the NBAIR BtPl was considered for whole genome sequencing to identify the cry genes present in it. Whole genome sequencing of our strain revealed genome size of 6.87Mb with 34.95% GC content. Homology search through the BLAST algorithm revealed that NBAIR BtPl is 99.8% similar to B. thuringiensis serovar tolworthi, and gene prediction through Prokka revealed 7406 genes, 7168 proteins, 5 rRNAs, and 66 tRNAs. BtToxin_Digger analysis of NBAIR BtPl genome revealed four cry gene families: cry1, cry2, cry8Aa1, and cry70Aa1. When tested for the presence of these four cry genes in other indigenous strains, results showed that cry70Aa1 was absent. Thus, the study provided a basis for predicting cry70Aa1 be the possible reason for toxicity. In this study apart from novel genes, we also identified other virulent genes encoding zwittermicin, chitinase, fengycin, and bacillibactin. Thus, the current study aids in predicting potential toxin-encoding genes responsible for toxicity to Z. cucurbitae and thus paves the way for the development of B. thuringiensis-based formulations and transgenic crops for management of dipteran pests.

Screening of indigenous Bacillus thuringiensis for dipteran active cry gene profiles and potential toxicity against melon fruit fly, Zeugodacus cucurbitae (Coquillett)

BackgroundMelon fruit fly, Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae), is a devastating polyphagous pest attacking large number of fruits and vegetables causing huge economic yield losses across the world. Management of this notorious pest is very challenging as the larvae feed inside the fruit. Hence, the present research study aimed to screen the indigenous Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) strains causing toxicity to larvae and to identify the insecticidal toxicity-related genes present in respective strains. In the present study, 50 indigenous B. thuringiensis (Bt) strains along with one reference strain were screened against second-instar larvae Z. cucurbitae. All the strains were analyzed for presence of 21 dipteran active cry genes.ResultsMortality in Z. cucurbitae larvae due to Bt strains ranged from 16 to 92%. PCR results revealed that each strain tested positive for a minimum of three cry genes to maximum of nine cry genes. Among the cry genes, cry1A, cry2A, cry1C, cry19, cry11 and cry70 were detected in high frequency of 100, 88, 84, 74, 58 and 54%, respectively. Bioassay studies revealed that ten out of fifty strains displayed more than 50% mortality. Hence, these ten strains, along with the reference strain, were further tested for mortality for the calculation of the median lethal concentration (LC50). The LC50 values ranged between 38.48 and 105.18 μg/ml. The lowest LC50 found for the strain NBAIR Bt107 was 38.48 μg/ml and was on par with the reference strain (Bti 4Q1) (31.3 μg/ml).ConclusionIndigenous Bt strains displayed a toxicity against the larvae of Z. cucurbitae. The probable dipteran active cry genes responsible for toxicity were interpreted. Thus, the Cry toxins from Bt can play a very important role in the management of Z. cucurbitae.

Association between PER and CRY gene polymorphisms and the response to caffeine citrate treatment in infants with apnea of prematurity.

Circadian rhythms impact metabolism and the therapeutic effects of drugs. The purpose of this study was to determine the association between PER and CRY polymorphisms and caffeine citrate treatment response in infants with apnea of prematurity. A total of 221 preterm infants of gestational age <34 weeks were included in this study (160 in the response group and 61 in the non-response group). The propensity score matching method was used to perform a 1:1 matching for all premature infants, and the general characteristics and clinical outcomes of the two groups were compared. The association between polymorphisms of the circadian transcription repressors PER and CRY and caffeine citrate treatment response in infants with apnea of prematurity was analyzed with co-dominant, dominant, recessive, and over-dominant models, as well as analysis of alleles. Generalized multifactor dimensionality reduction (GMDR) analysis was used to analyze the interaction between the PER and CRY genes. After propensity score matching, 45 preterm infants were included in each of the response and non-response groups, and there were no statistically significant differences in general characteristics between the two groups (P > 0.05). Infants in the non-response groups had a higher incidence of moderate and severe bronchopulmonary dysplasia (BPD) (P = 0.043), retinopathy of prematurity (ROP) (P = 0.035), and invasive ventilation (P = 0.027), and their duration of oxygen use (P = 0.041) was longer. When corrected for false discovery rate, the PER3 rs228669 recessive model (P FDR = 0.045) and the over-dominant model (P FDR = 0.045) were both associated with caffeine citrate treatment response. Preterm infants with the rs228669 CC genotype had a significantly lower rate of caffeine citrate non-response in the recessive model (OR = 0.28, 95% CI = 0.12-0.66), which was significantly higher in preterm infants with the CT genotype in the over-dominant model (OR = 4.18, 95% CI = 1.64-10.66). GMDR analysis revealed an interaction between the PER and CRY genes (P < 0.05). Circadian rhythms may play a role in the response of premature infants to caffeine citrate, and polymorphisms of the PER and CRY genes may influence the effectiveness of caffeine citrate treatment for apnea of prematurity.

Molecular Characterisation and Toxicity Analysis of Indigenous Bacillus thuringiensis Berliner Isolates against Cucurbit Fruit Fly Maggots, Zeugodacus cucurbitae (Coquilett) (Diptera: Tephritidae)

Background: Indiscriminate use of insecticides against fruit flies (Tephritidae: Diptera) has led to the development of residue and resistance and there is a need to try alternate eco-friendly management practices. Bacillus thuringiensis (Bt) is the most widely used bacterium forming proteins active against insects of different orders. Thus, the present study was framed to characterize and evaluate 50 indigenous Bt isolates against cucurbit fruit fly maggots, Zeugodacus cucurbitae. Methods: Colony morphology was observed visually, while crystal morphology was observed through a microscope. The cry gene content of the Bt isolates was screened by Polymerase Chain Reaction and the proteins harbored were assessed by SDS- PAGE analysis. Full diet contamination method was used to evaluate the toxicity of Bt isolates against cucurbit fruit fly maggots. Result: Uniform full white, off-white and creamy white colony colours were found except one isolate each, exhibiting full white at centre surrounded by off white and off white at centre surrounded by full white. Spherical shape crystal (45.10%) was predominant followed by cuboidal (29.41%), bipyramidal (17.65%), rectangular (3.92%) and minute crystal attached to spore (3.92%). Insecticidal proteins varied from ~15 to greater than 200kDa in size with one to four and more distinct bands per isolate. PCR screening revealed cry4Aa in 2 isolates, cry4Ba and cry11Aa in one isolate each, cyt1 in 1 isolate. The standard strain Bti 4Q2 (100% mortality of maggots) and the indigenous Bt isolates, T166 (92.59%), T184 (91.11%) and T60 (90.48%) were found to be significantly toxic.

Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study

BackgroundAs part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants.ResultsTo assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves.ConclusionsOur work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

Bifidobacterium adolescentis-derived hypaphorine alleviates acetaminophen hepatotoxicity by promoting hepatic Cry1 expression

Acetaminophen (APAP)-induced liver injury (AILI) is a pressing public health concern. Although evidence suggests that Bifidobacterium adolescentis (B. adolescentis) can be used to treat liver disease, it is unclear if it can prevent AILI. In this report, we prove that B. adolescentis significantly attenuated AILI in mice, as demonstrated through biochemical analysis, histopathology, and enzyme-linked immunosorbent assays. Based on untargeted metabolomics and in vitro cultures, we found that B. adolescentis generates microbial metabolite hypaphorine. Functionally, hypaphorine inhibits the inflammatory response and hepatic oxidative stress to alleviate AILI in mice. Transcriptomic analysis indicates that Cry1 expression is increased in APAP-treated mice after hypaphorine treatment. Overexpression of Cry1 by its stabilizer KL001 effectively mitigates liver damage arising from oxidative stress in APAP-treated mice. Using the gene expression omnibus (GEO) database, we verified that Cry1 gene expression was also decreased in patients with APAP-induced acute liver failure. In conclusion, this study demonstrates that B. adolescentis inhibits APAP-induced liver injury by generating hypaphorine, which subsequently upregulates Cry1 to decrease inflammation and oxidative stress.

Dietary Oat β-Glucan Alleviates High-Fat Induced Insulin Resistance through Regulating Circadian Clock and Gut Microbiome.

High-fat diet induced circadian rhythm disorders (CRD) are associated with metabolic diseases. As the main functional bioactive component in oat, β-glucan (GLU) can improve metabolic disorders, however its regulatory effect on CRD remains unclear. In this research, the effects of GLU on high-fat diet induced insulin resistance and its mechanisms are investigated, especially focusing on circadian rhythm-related process. Male C57BL/6 mice are fed a low fat diet, a high-fat diet (HFD), and HFD supplemented 3% GLU for 13 weeks. The results show that GLU treatment alleviates HFD-induced insulin resistance and intestinal barrier dysfunction in obese mice. The rhythmic expressions of circadian clock genes (Bmal1, Clock, and Cry1) in the colon impaired by HFD diet are also restored by GLU. Further analysis shows that GLU treatment restores the oscillatory nature of gut microbiome, which can enhance glucagon-like peptide (GLP-1) secretion via short-chain fatty acids (SCFAs) mediated activation of G protein-coupled receptors (GPCRs). Meanwhile, GLU consumption significantly relieves colonic inflammation and insulin resistance through modulating HDAC3/NF-κB signaling pathway. GLU can ameliorate insulin resistance due to its regulation of colonic circadian clock and gut microbiome.

Role for clock protein PER1 in cardiac adaption to salt-sensitive hypertension in male rats

The clock protein PERIOD1 (PER1) is known to regulate the transcription of many genes, impacting subsequent physiological responses and working with other proteins to establish rhythmicity in processes such as blood pressure regulation. Global PER1 knock-out (KO) in male rats is associated with increased severity of the salt-sensitive (SS) hypertensive phenotype in Dahl SS rats. Male PER1 KO SS rats demonstrated reduced amplitude of heart rate as well as altered renal electrolyte and fluid balance with accompanying changes in the expression of relevant genes in the kidney. However, little is known regarding changes in gene expression in cardiac tissue which may contribute to this phenotype. The goal of this study was to test the hypothesis that knockout of PER1 in SS rats results in altered gene expression in the heart. We selected the following genes due to their role in the clock protein complex and circadian control over blood pressure: Basic Helix-Loop-Helix ARNT Like 1 ( Bmal1), Cryptochrome 1 and 2 ( Cry1, Cry2), and Period 2 ( Per2). Given its role in compensatory natriuretic responses to pressure overload, we also assessed the gene Nppa which encodes atrial natriuretic peptide (ANP). In order to test this hypothesis, hearts were collected at 2 am (during the rat active period) from male Dahl SS control and PER1 KO rats following 3 weeks of high salt (4% NaCl) diet (n=5-6). Changes in expression of candidate genes were measured using quantitative real time RT-PCR. Genotype effects were assessed using unpaired student’s t test. PER1 KO SS male rats on high salt diet exhibited significantly decreased cardiac expression of in the clock genes Bmal1 (p&lt;0.0001), Cry1 (p=0.0007), and Cry2 (p=0.0094). Changes in Per2 expression were not apparent (p=0.1152). In contrast, PER1 KO rats exhibited significantly increased expression of Nppa (p=0.0017). Global PER1 knock-out in male SS rats results in hypertension associated with disrupted rhythms in heart rate. These changes are associated with reduction in expression of the genes encoding regulatory clock proteins Bmal1, Cry1, and Cry2, but not Per2. The decreased circadian gene expression in PER1 KO Dahl SS rats may contribute to the disrupted rhythms in heart rate. Increased ANP gene expression in the hearts of PER1 KO SS rats could serve as a compensatory mechanism to induce natriuresis. Thus, the data support the hypothesis that knockout of PER1 in SS rats alters gene expression in the heart. Together, these data illuminate a possible circadian-regulated pathway through which the heart and kidney communicate to respond to fluid and electrolyte imbalance in salt-sensitive hypertension. This work was supported by the National Institutes of Health grants R35 HL135789 (to AS), R01 DK109570 (to MLG and AS), R56DK128271 (to MLG), and Department of Veteran Affairs grants I01 BX004024 (to AS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Assessing the Feasibility and Efficacy of Pre-Sleep Dim Light Therapy for Adults with Insomnia: A Pilot Study.

Background: Insomnia is increasingly recognized for its marked impact on public health and is often associated with various adverse health outcomes, including cardiovascular diseases and mental health disorders. The aim of this study was to investigate the efficacy of pre-sleep dim light therapy (LT) as a non-pharmacological intervention for insomnia in adults, assessing its influence on sleep parameters and circadian rhythms. Methods: A randomized, open-label, two-arm clinical trial was conducted over two weeks with 40 participants aged 20-60 years, all of whom had sleep disorders (CRIS, KCT0008501). They were allocated into control and LT groups. The LT group received exposure to warm-colored light, minimizing the blue spectrum, before bedtime. The study combined subjective evaluation via validated, sleep-related questionnaires, objective sleep assessments via actigraphy, and molecular analyses of circadian clock gene expression in peripheral blood mononuclear cells. Baseline characteristics between the two groups were compared using an independent t-test for continuous variables and the chi-squared test for categorical variables. Within-group differences were assessed using the paired t-test. Changes between groups were analyzed using linear regression, adjusting for each baseline value and body mass index. The patterns of changes in sleep parameters were calculated using a linear mixed model. Results: The LT group exhibited significant improvements in sleep quality (difference in difference [95% CI]; -2.00 [-3.58, -0.43], and sleep efficiency (LT: 84.98 vs. control: 82.11, p = 0.032), and an advanced Dim Light Melatonin Onset compared to the control group (approximately 30 min). Molecular analysis indicated a significant reduction in CRY1 gene expression after LT, suggesting an influence on circadian signals for sleep regulation. Conclusions: This study provides evidence for the efficacy of LT in improving sleep quality and circadian rhythm alignment in adults with insomnia. Despite limitations, such as a small sample size and short study duration, the results underscore the potential of LT as a viable non-pharmacological approach for insomnia. Future research should expand on these results with larger and more diverse cohorts followed over a longer period to validate and further elucidate the value of LT in sleep medicine. Trial registration: The trial was registered with the Clinical Research Information Service (KCT0008501).

OpsinLW2 serves as a circadian photoreceptor in the entrainment of circadian locomotor rhythm of a firebrat

Photic entrainment is an essential function of the circadian clock, which enables organisms to set the appropriate timing of daily behavioral and physiological events. Recent studies have shown that the mechanisms of the circadian clock and photic entrainment vary among insect species. This study aimed to elucidate the circadian photoreceptors necessary for photic entrainment in firebrats Thermobia domestica, one of the most primitive apterygote insects. A homology search of publicly available RNA sequence (RNA-seq) data from T. domestica exhibited a cryptochrome 2 (cry2) gene and three opsin genes, opsin long wavelength 1 (opLW1), opLW2, and opUV, as candidate circadian photoreceptors. We examined the possible involvement of these genes in photic entrainment of firebrat locomotor rhythms. Firebrats had the highest entrainability to the light–dark cycle of green light. Treatment with dsRNA of the candidate genes strongly downregulated the respective targeted genes, and in the case of opsin genes, other untargeted genes were occasionally downregulated to various degrees. Under constant light, most control firebrats became arrhythmic, whereas a fraction of those treated with double RNAi of the two opLWs remained rhythmic. Behavioral experiments revealed that the transient cycles necessary for re-entrainment to shifted light cycles were lengthened when opLW2 expression was reduced. These results suggest that opLW2 is involved in the photic entrainment of circadian rhythm in firebrats.

Chronic sleep fragmentation reduces left ventricular contractile function and alters gene expression related to innate immune response and circadian rhythm in the mouse heart

Sleep disorders have emerged as a widespread public health concern, primarily due to their association with an increased risk of developing cardiovascular diseases. Our previous research indicated a potential direct impact of insufficient sleep duration on cardiac remodeling in children and adolescents. Nevertheless, the underlying mechanisms behind the link between sleep fragmentation (SF) and cardiac abnormalities remain unclear. In this study, we aimed to investigate the effects of SF interventions at various life stages on cardiac structure and function, as well as to identify genes associated with SF-induced cardiac dysfunction. To achieve this, we established mouse models of chronic SF and two-week sleep recovery (SR). Our results revealed that chronic SF significantly compromised left ventricular contractile function across different life stages, leading to alterations in cardiac structure and ventricular remodeling, particularly during early life stages. Moreover, microarray analysis of mouse heart tissue identified two significant modules and nine hub genes (Ddx60, Irf9, Oasl2, Rnf213, Cmpk2, Stat2, Parp14, Gbp3, and Herc6) through protein–protein interaction analysis. Notably, the interactome predominantly involved innate immune responses. Importantly, all hub genes lost significance following SR. The second module primarily consisted of circadian clock genes, and real-time PCR validation demonstrated significant upregulation of Arntl, Dbp, and Cry1 after SF, while subsequent SR restored normal Arntl expression. Furthermore, the expression levels of four hub genes (Ddx60, Irf9, Oasl2, and Cmpk2) and three circadian clock genes (Arntl, Dbp, and Cry1) exhibited correlations with structural and functional echocardiographic parameters. Overall, our findings suggest that SF impairs left ventricular contractile function and ventricular remodeling during early life stages, and this may be mediated by modulation of the innate immune response and circadian rhythm. Importantly, our findings suggest that a short period of SR can alleviate the detrimental effects of SF on the cardiac immune response, while the influence of SF on circadian rhythm appears to be more persistent. These findings underscore the importance of good sleep for maintaining cardiac health, particularly during early life stages.

Functional Analyses of Four Cryptochromes From Aquatic Organisms After Heterologous Expression in Drosophila melanogaster Circadian Clock Cells.

Cryptochromes (Crys) represent a multi-facetted class of proteins closely associated with circadian clocks. They have been shown to function as photoreceptors but also to fulfill light-independent roles as transcriptional repressors within the negative feedback loop of the circadian clock. In addition, there is evidence for Crys being involved in light-dependent magneto-sensing, and regulation of neuronal activity in insects, adding to the functional diversity of this cryptic protein class. In mammals, Crys are essential components of the circadian clock, but their role in other vertebrates is less clear. In invertebrates, Crys can function as circadian photoreceptors, or as components of the circadian clock, while in some species, both light-receptive and clock factor roles coexist. In the current study, we investigate the function of Cry proteins in zebrafish (Danio rerio), a freshwater teleost expressing 6 cry genes. Zebrafish peripheral circadian clocks are intrinsically light-sensitive, suggesting the involvement of Cry in light-resetting. Echinoderms (Strongylocentrotus purpuratus) represent the only class of deuterostomes that possess an orthologue (SpuCry) of the light-sensitive Drosophila melanogaster Cry, which is an important component of the light-resetting pathway, but also works as transcriptional repressor in peripheral clocks of fruit flies. We therefore investigated the potential of different zebrafish cry genes and SpuCry to replace the light-resetting and repressor functions of Drosophila Cry by expressing them in fruit flies lacking endogenous cry function. Using various behavioral and molecular approaches, we show that most Cry proteins analyzed are able to fulfill circadian repressor functions in flies, except for one of the zebrafish Crys, encoded by cry4a. Cry4a also shows a tendency to support light-dependent Cry functions, indicating that it might act in the light-input pathway of zebrafish.