Genomic and Epigenomic Changes in the Progeny of Cold-Stressed Arabidopsis thaliana Plants.

Plants are continuously exposed to various environmental stresses. Because they can not escape stress, they have to develop mechanisms of remembering stress exposures somatically and passing it to the progeny. We studied the Arabidopsis thaliana ecotype Columbia plants exposed to cold stress for 25 continuous generations. Our study revealed that multigenerational exposure to cold stress resulted in the changes in the genome and epigenome (DNA methylation) across generations. Main changes in the progeny were due to the high frequency of genetic mutations rather than epigenetic changes; the difference was primarily in single nucleotide substitutions and deletions. The progeny of cold-stressed plants exhibited the higher rate of missense non-synonymous mutations as compared to the progeny of control plants. At the same time, epigenetic changes were more common in the CHG (C = cytosine, H = cytosine, adenine or thymine, G = guanine) and CHH contexts and favored hypomethylation. There was an increase in the frequency of C to T (thymine) transitions at the CHH positions in the progeny of cold stressed plants; because this type of mutations is often due to the deamination of the methylated cytosines, it can be hypothesized that environment-induced changes in methylation contribute to mutagenesis and may be to microevolution processes and that RNA-dependent DNA methylation plays a crucial role. Our work supports the existence of heritable stress response in plants and demonstrates that genetic changes prevail.

A National Perspective on the Impact of the COVID-19 Pandemic on Heart Failure Hospitalizations in the United States.

Nationwide data of the COVID-19 pandemic's impact on heart failure (HF) hospitalizations is lacking. We conducted this study to elucidate the impact of the COVID-19 pandemic on HF hospitalizations. Additionally, we assessed the differences in hospitalization characteristics during the pandemic and the impact that a concurrent diagnosis of COVID-19 has on various outcomes and predictors of inpatient mortality among patients admitted for HF. The National Inpatient Sample (NIS) database was queried for all hospitalizations with a primary diagnosis of HF between 2017 and 2020. Monthly HF hospitalizations were trended longitudinally over this period. Beginning April 1, 2020, concurrent COVID-19 infections were identified. Subsequently, we stratified HF hospitalizations between April 2020 and December 2020 (HF-2020) based on if concomitant COVID-19 was diagnosed, forming the HF-COVID+ve and HF-COVID-ve groups respectively. HF-2020 was also compared with prepandemic HF hospitalizations between April 2019 and December 2019 (HF-2019). Baseline characteristics were compared, and adjusted outcomes were obtained. During the initial COVID-19 surge in April 2020, HF admissions were reduced by 47% compared to January 2020. Following this decline, HF hospitalizations increased but did not reach prepandemic levels. HF-2020 admissions had an increased complication burden compared to HF-2019, including acute myocardial infarction (8.9% vs 6.6%, P < 0.005) and pulmonary embolism (4.1% vs 3.4%, P < 0.005) indicating a sicker cohort of patients. HF-COVID+ve hospitalizations had 2.9 times higher odds of inpatient mortality compared to HF-COVID-ve and an increased adjusted length of stay by 2.16 days (P < 0.005). A pandemic of the same magnitude as COVID-19 can overwhelm even the most advanced health systems. Early resource mobilization and preparedness is essential to provide care to a sick cohort of patients like acute HF, who are directly and indirectly effected by the consequences of the pandemic which has worsened hospitalization outcomes.

Combined immunotherapeutic effect of Leishmania-derived recombinant aldolase and Ambisome against experimental visceral leishmaniasis.

BACKGROUND: Available therapeutics for visceral leishmaniasis (VL), a deadly parasitic infection, are usually associated with inadequate efficacy and adverse aftereffects. Further, the primary site of Leishmania parasite are host macrophages resulting in compromised immunity; ensuing marked T-cell immunosuppression. Such settings emphasize the exploration of chemo-immunotherapeutic strategies for improvising the infected person's immune status with better resolution of infection. METHODS: Present work employs the immunization of Leishmania-infected hamsters with Leishmania-derived recombinant aldolase (rLdAld) and enolase (rLdEno) proteins in consort with the sub-optimal dose of Ambisome (2.5 mg/kg). After the completion of immunization, hamsters were sacrificed on day 60 and 90 post infection and different organ samples were collected to perform immunological assay for evaluating the therapeutic efficacy and modulation in protective cellular immune responses. RESULTS: Combining these proteins, particularly rLdAld with Ambisome (2.5 mg/kg), has significantly reduced the parasitic load (∼80%) with remarkable enhancement in DTH and lymphoproliferative responses compared to the infected control and only Ambisome treated groups. Moreover, cytokine levels at RNA and protein levels were noticed to be inclined towards Th-1 phenotype through up-regulation of IFN-γ and TNF-α with significant down-regulation in IL-10 and TGF-ß expression, an indication towards the generation of protective immunity against experimental VL. CONCLUSION: Our experimental findings demonstrated that the chemo-immunotherapeutic approach could be an effective way of controlling human VL infection at minimal dosages of antileishmanial with reduced side-effects and propensity of drug resistance emergence.

Gut Microbiota Interventions for the Management of Obesity: A Literature Review.

The gut microbiota (GM) has been recognized as an important factor in the development of metabolic diseases such as obesity; it has been reported that the composition of the GM differs in obese and lean subjects, suggesting that microbiota dysbiosis can contribute to changes in body weight. Dysbiosis occurs due to an imbalance in the composition of gut bacteria, changes in the metabolic process, or changes in the distribution of microbiota within the gut. Dysbiosis can change the functioning of the intestinal barrier and the gut-associated lymphoid tissues (GALT). Microbial manipulation may help with preventing or treating weight gain and associated comorbidities. Approaches to this may range from dietary manipulation, which is suitable to treat the individual's microflora, to probiotics, prebiotics, synbiotics, and fecal microbiota transplant (FMT).

Paclobutrazol improves surface water use efficiency by regulating allometric trait behavior in maize.

Paclobutrazol (PBZ) role in drought management of maize is least understood. In maize, root traits are linked with surface water management. Over three years, early and terminal deficit irrigation (EDI and TDI) with or without PBZ were imposed on DKC-9144 and PG-2475 maize varieties. Several allometric parameters viz. stem height, stem diameter, leaf area and root traits along with physiological processes were measured. Implication of these parameters in the management of soil surface irrigation in terms of water use efficiency (WUE) was demonstrated in maize. Increased number of lateral roots and root number density in DKC-9144 provided more surface area for water absorption for better management of EDI. Root growth rates showed a similar pattern with root length, root surface areas, and root numbers in EDI. Elevated expressions of ZmRTCL, ZmRTCS and ZmARF34 in EDI and EDI plus PBZ were associated with seminal roots and root laterals initiation. Under TDI alone or in combination with PBZ, root lengths (BRL, CRL, SRL) and root surface areas varied in DKC-9144 and PG-2475 over control. Furthermore, correlation analysis showed that decrease in WUE under TDI was significantly associated with a reduction in stem thickness and leaf surface area. For WUE_N in TDI and PBZ plus TDI, structural equation modelling proposed, brace root surface area (BRSA_N) as a positive contributor, while a negative contributor was seminal root surface area (SRSA_N). Present study explained the importance of specific root traits and their association with other allometric parameters for improving WUE in DKC-9144 variety of maize and the crop in general.

Multigenerational Exposure to Heat Stress Induces Phenotypic Resilience, and Genetic and Epigenetic Variations in Arabidopsis thaliana Offspring.

Plants are sedentary organisms that constantly sense changes in their environment and react to various environmental cues. On a short-time scale, plants respond through alterations in their physiology, and on a long-time scale, plants alter their development and pass on the memory of stress to the progeny. The latter is controlled genetically and epigenetically and allows the progeny to be primed for future stress encounters, thus increasing the likelihood of survival. The current study intended to explore the effects of multigenerational heat stress in Arabidopsis thaliana. Twenty-five generations of Arabidopsis thaliana were propagated in the presence of heat stress. The multigenerational stressed lineage F25H exhibited a higher tolerance to heat stress and elevated frequency of homologous recombination, as compared to the parallel control progeny F25C. A comparison of genomic sequences revealed that the F25H lineage had a three-fold higher number of mutations [single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs)] as compared control lineages, suggesting that heat stress induced genetic variations in the heat-stressed progeny. The F25H stressed progeny showed a 7-fold higher number of non-synonymous mutations than the F25C line. Methylome analysis revealed that the F25H stressed progeny showed a lower global methylation level in the CHH context than the control progeny. The F25H and F25C lineages were different from the parental control lineage F2C by 66,491 and 80,464 differentially methylated positions (DMPs), respectively. F25H stressed progeny displayed higher frequency of methylation changes in the gene body and lower in the body of transposable elements (TEs). Gene Ontology analysis revealed that CG-DMRs were enriched in processes such as response to abiotic and biotic stimulus, cell organizations and biogenesis, and DNA or RNA metabolism. Hierarchical clustering of these epimutations separated the heat stressed and control parental progenies into distinct groups which revealed the non-random nature of epimutations. We observed an overall higher number of epigenetic variations than genetic variations in all comparison groups, indicating that epigenetic variations are more prevalent than genetic variations. The largest difference in epigenetic and genetic variations was observed between control plants comparison (F25C vs. F2C), which clearly indicated that the spontaneous nature of epigenetic variations and heat-inducible nature of genetic variations. Overall, our study showed that progenies derived from multigenerational heat stress displayed a notable adaption in context of phenotypic, genotypic and epigenotypic resilience.

Compliance with the smoke-free public places legislation in Nepal: A cross-sectional study from Biratnagar Metropolitan City.

BACKGROUND: Smoke-free legislation banning tobacco smoking in public places was implemented across Nepal in 2014 with the ambition to reduce the impact of second-hand smoking. As part of a comprehensive policy package on tobacco control, the implementation of the legislation has seen a marked reduction in tobacco consumption. Yet there remains uncertainty about the level of compliance with smoke-free public places. OBJECTIVES: This study assesses the compliance with smoke-free laws in public places and the factors associated with active smoking in public places in Biratnagar Metropolitan City, Nepal. METHODS: A cross-sectional study was conducted in the Biratnagar metropolitan city in Province 1 of Nepal from July to December 2019. A total of 725 public places within the metropolitan city were surveyed using a structured survey tool. Active smoking was the primary outcome of the study which was defined as smoking by any person during the data collection time at the designated public place. RESULTS: The overall compliance with smoke-free legislation was 56.4%. The highest compliance (75.0%) was observed in Government office buildings. The lowest compliance was observed in eateries, entertainment, and shopping venues (26.3%). There was a statistically significant association between active smoking and the presence of 'no smoking' notices appended at the entrance and the odds of active smoking in eateries, entertainment, hospitality, shopping venues, transportations and transits was higher compared to education and health care institutions. None of the 'no smoking' notices displayed fully adhered to the contents as prescribed by the law. CONCLUSION: As more than half of the public places complied with the requirements of the legislation, there was satisfactory overall compliance with the smoke-free public places law in this study. The public venues (eateries, shopping venues and transportations) that are more frequently visited and have a high turnover of the public have lower compliance with the legislation. The content of the message in the 'no smoking' notices needs close attention to adhere to the legal requirements.

Nanoformulations of quercetin: a potential phytochemical for the treatment of uv radiation induced skin damages

Abstract The continuous prolonged exposures of sun light especially the ultra violet (UV) radiation present in it, cause not only the risk of skin cancer but also it may cause premature skin aging, photodermatoses and actinic keratoses. Flavonoids (including Flavane, Flavanone, Flavone, Flavonol, Isoflavone, Neoflavone etc.) having potent antioxidant activity, used as topical applications for protection against UV induced skin damages as well as for skin care. Most commonly used flavonoid is quercetin (Flavonol), which is present in fruits, vegetables, and herbs. We aim to review the research focused on development of different novel formulations to treat UV radiations induced skin diseases. In this review, several formulations of flavonoid quercetin were discussed and their outcomes were compiled and compared in context to solubility, stability and efficiency of application. On the basis this comparative analysis we have concluded that three formulations, namely glycerosomes, nanostructured lipid carriers and deformable liposomes hold good applications for future aspects for topical delivery of quercetin. These formulations showed enhanced stability, increased quercetin accumulation in different skin layers, facilitated drug permeation in skin and long-lasting drug release.

Special Issue Editorial: Isolation and Analysis of Characteristic Compounds from Herbal and Plant Extracts.

Herbal and plant extracts exhibit various types of properties and activities that have been applied in the medicinal field to treat diseases and achieve better health [...].

Plants Metabolome Study: Emerging Tools and Techniques.

Metabolomics is now considered a wide-ranging, sensitive and practical approach to acquire useful information on the composition of a metabolite pool present in any organism, including plants. Investigating metabolomic regulation in plants is essential to understand their adaptation, acclimation and defense responses to environmental stresses through the production of numerous metabolites. Moreover, metabolomics can be easily applied for the phenotyping of plants; and thus, it has great potential to be used in genome editing programs to develop superior next-generation crops. This review describes the recent analytical tools and techniques available to study plants metabolome, along with their significance of sample preparation using targeted and non-targeted methods. Advanced analytical tools, like gas chromatography-mass spectrometry (GC-MS), liquid chromatography mass-spectroscopy (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) matrix-assisted laser desorption/ionization (MALDI), ion mobility spectrometry (IMS) and nuclear magnetic resonance (NMR) have speed up precise metabolic profiling in plants. Further, we provide a complete overview of bioinformatics tools and plant metabolome database that can be utilized to advance our knowledge to plant biology.

OMICs, Epigenetics, and Genome Editing Techniques for Food and Nutritional Security.

The incredible success of crop breeding and agricultural innovation in the last century greatly contributed to the Green Revolution, which significantly increased yields and ensures food security, despite the population explosion. However, new challenges such as rapid climate change, deteriorating soil, and the accumulation of pollutants require much faster responses and more effective solutions that cannot be achieved through traditional breeding. Further prospects for increasing the efficiency of agriculture are undoubtedly associated with the inclusion in the breeding strategy of new knowledge obtained using high-throughput technologies and new tools in the future to ensure the design of new plant genomes and predict the desired phenotype. This article provides an overview of the current state of research in these areas, as well as the study of soil and plant microbiomes, and the prospective use of their potential in a new field of microbiome engineering. In terms of genomic and phenomic predictions, we also propose an integrated approach that combines high-density genotyping and high-throughput phenotyping techniques, which can improve the prediction accuracy of quantitative traits in crop species.

A Chimera of Th1 Stimulatory Proteins of Leishmania donovani Offers Moderate Immunotherapeutic Efficacy with a Th1-Inclined Immune Response against Visceral Leishmaniasis.

Immunotherapy, a treatment based on host immune system activation, has been shown to provide a substitute for marginally effective conventional chemotherapy in controlling visceral leishmaniasis (VL), the deadliest form of leishmaniasis. As the majority of endemic inhabitants exhibit either subclinical or asymptomatic infection which often develops into the active disease state, therapeutic intervention seems to be an important avenue for combating infections by stimulating the natural defense system of infected individuals. With this perspective, the present study focuses on two immunodominant Leishmania (L.) donovani antigens (triosephosphate isomerase and enolase) previously proved to be potent prophylactic VL vaccine candidates, for generating a recombinant chimeric antigen. This is based on the premise that in a heterogeneous population, a multivalent antigen vaccine would be required for an effective response against leishmaniasis (a complex parasitic disease). The resulting molecule rLdT-E chimeric protein was evaluated for its immunogenicity and immunotherapeutic efficacy. A Th1 stimulating adjuvant BCG was employed with the protein which showed a remarkable 70% inhibition of splenic parasitic multiplication positively correlated with boosted Th1 dominant immune response against lethal L. donovani challenge in hamsters as evidenced by high IFN-γ and TNF-α and low IL-10. In addition, immunological analysis of antibody subclass presented IgG2-based humoral response besides considerable delayed-type hypersensitivity and lymphocyte proliferative responses in rLdT-E/BCG-treated animals. Our observations indicate the potential of the chimera towards its candidature for an effective vaccine against Leishmania donovani infection.

Development and Standardization of Rapid and Efficient Seed Germination Protocol for Cannabis sativa.

Cannabis seed germination is an important process for growers and researchers alike. Many biotechnological applications require a reliable sterile method for seed germination. This protocol outlines a seed germination procedure for Cannabis sativa using a hydrogen peroxide (H2O2) solution as liquid germination media. In this protocol, all three steps including seed sterilization, germination, and seedlings development were carried out in an H2O2 solution of different concentrations; 1% H2O2 solution showed the fastest and the most efficient germination. This protocol also exhibited high germination efficiency for very old cannabis seeds with lower viability. Overall, this protocol demonstrates superior germination compared to water control and reduces the risk of contamination, making it suitable for tissue culture and other sensitive applications.

Nickel excess affects phenology and reproductive attributes of Asterella wallichiana and Plagiochasma appendiculatum growing in natural habitats.

Bryophytes are potent metal absorbers, thriving well on heavy metal (HM)-polluted soils. Mechanisms controlling uptake, compartmentalization and impacts of HMs on bryophytes life cycle are largely unknown. The current study is an effort to decipher mechanisms of nickel (Ni) excess-induced effects on the phenological events of two bryophytes, Asterella wallichiana and Plagiochasma apendiculatum growing in natural habitats. Observations revealed Ni-excess induced negative impacts on abundance, frequency of occurrence of reproductive organs, population viability and morphological traits, spore viability and physiological attributes of both the liverworts. Results led us conclude that P. appendiculatum survived better with the lowest impact on its life cycle events than A. wallichiana under Ni excess in natural habitats. Our findings collectively provide insights into the previously unknown mechanisms of Ni-induced responses in liverworts with respect to phenological attributes, as well as demonstrate the potential of P. appendiculatum to survive better in Ni excess habitats.

Erdheim Chester disease: a subtle quiddity; the first case reported from Nepal.

Erdheim-Chester disease (ECD) is a rare entity throughout the world. This is the first case reported in Nepal. ECD is a rare aggressive, non-Langerhan's histocytosis of unknown origin with classical histological features. The patient usually presents with bone pain or skeletal symptoms along with other constitutional syndrome. Although, no definitive therapy has been approved, interferon-alfa (or Pegylated Interferon-alfa) is considered as initial therapy. In this case report, we found a patient with right-sided localized chest pain for which he was evaluated with bone scan and excisional bone biopsy and its findings support the diagnosis of ECD.

Transient expression of the ß-glucuronidase gene in Cannabis sativa varieties.

In plant biology, transient expression analysis plays a vital role to provide a fast method to study the gene of interest. In this study, we report a rapid and efficient method for transient expression in Cannabis sativa seedlings using Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 carrying the pCAMBIA1301 construct with uidA gene was used to transform cannabis seedlings and the GUS assay (a measurement of ß-glucuronidase activity) was used to detect the uidA expression. In the current study, we have also established a rapid germination protocol for cannabis seeds. The all three steps seed sterilization, germination and seedlings development were carried out in a 1% H2O2 solution. Transient transformation revealed that both cotyledons and young true leaves are amenable to transformation. Compared with tobacco (Nicotiana benthamiana), cannabis seedlings were less susceptible to transformation with A. tumefaciens. Susceptibility to Agrobacterium transformation also varied with the different cannabis varieties. The method established in this study has the potential to be an important tool for gene function studies and genetic improvement in cannabis.

Development and Optimization of a Germination Assay and Long-Term Storage for Cannabis sativa Pollen.

Pollen viability and storage is of great interest to cannabis breeders and researchers to maintain desirable germplasm for future use in breeding or for biotechnological and gene editing applications. Here, we report a simple and efficient cryopreservation method for long-term storage of Cannabis sativa pollen. Additionally, the bicellular nature of cannabis pollen was identified using DAPI (4',6-diamidino-2-phenylindole) staining. A pollen germination assay was developed to assess cannabis pollen viability and used to demonstrate that pollen collected from different principal growth stages exhibited differential longevity. Finally, a simple and efficient method that employs pollen combined with baked whole wheat flour and subsequent desiccation under vacuum was developed for the long-term cryopreservation of C. sativa pollen. Using this method, pollen viability was maintained in liquid nitrogen after four months, suggesting long-term preservation of cannabis pollen.

Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts.

We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated "Edit Plasmids," were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions at one of the Cas9 cleavage sites in Chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated non-homologous end joining (NHEJ) repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.

Epigenetic aspects of floral homeotic genes in relation to sexual dimorphism in the dioecious plant Mercurialis annua.

In plants, dioecy characterizes species that carry male and female flowers on separate plants and it occurs in about 6% of angiosperms; however, the molecular mechanisms that underlie dioecy are essentially unknown. The ability for sex-reversal by hormone application raises the hypothesis that the genes required for the expression of both sexes are potentially functional but are regulated by epigenetic means. In this study, proteomic analysis of nuclear proteins isolated from flower buds of females, males, and feminized males of the dioecious plant Mercurialis annua revealed differential expression of nuclear proteins that are implicated in chromatin structure and function, including floral homeotic proteins. Focusing on floral genes, we found that class B genes were mainly expressed in male flowers, while class D genes, as well as SUPERMAN-like genes, were mainly expressed in female flowers. Cytokinin-induced feminization of male plants was associated with down-regulation of male-specific genes concomitantly with up-regulation of female-specific genes. No correlation was found between the expression of class B and D genes and the changes in DNA methylation or chromatin conformation of these genes. Thus, we could not confirm DNA methylation or chromatin conformation of floral genes to be the major determinant regulating sexual dimorphisms. Instead, determination of sex in M. annua might be controlled upstream of floral genes by one or more sex-specific factors that affect hormonal homeostasis. A comprehensive model is proposed for sex-determination in M. annua.

Post-intense exercise sleep concerns in weightlifters: A pilot study.

BACKGROUND: Weightlifting is a power and dynamic strength game. Performance of weightlifters during competition depends on absolute and relative power output achieved by them. To enhance power and strength in weightlifters, training programmes have frequent high-intensity exercise sessions. On an informal interaction with the sportspersons, they often complain of problems after intense training. There was a felt need to analyse the problems they perceive after intense training. METHOD: A feedback form was developed after brainstorming with coaches and sports medicine physicians of a sports institute. Thirty-six male weightlifters aged 19.78 ± 4.71 yrs (mean ± standard deviation) of an institute participated after obtaining informed consent. The feedback form was collected and analysed. RESULT: After an intense exercise, participants reported complaints such as increased fatigue (n = 27), sleep problems (n = 26), restlessness (n = 18) and decreased appetite (n = 12). Of the sleep problems, 69.4% (n = 25 out of 36) noticed worsening of sleep, 50% (n = 18) noticed sleep was light after intense training, 58.3% (n = 21) felt difficulty in falling asleep, 47.2% (n = 7) complained of frequent waking up during sleep at night and 77.8% (n = 28) felt sleepy after waking up. CONCLUSION: Intense training encounters certain problems such as fatigue, sleep disturbances, restlessness and decreased appetite.