Genetic manipulation of betta fish.

Betta splendens, also known as Siamese fighting fish or "betta," is a freshwater fish species renowned for its astonishing morphological diversity and extreme aggressive behavior. Despite recent advances in our understanding of the genetics and neurobiology of betta, the lack of tools to manipulate their genome has hindered progress at functional and mechanistic levels. In this study, we outline the use of three genetic manipulation technologies, which we have optimized for use in betta: CRISPR/Cas9-mediated knockout, CRISPR/Cas9-mediated knockin, and Tol2-mediated transgenesis. We knocked out three genes: alkal2l, bco1l, and mitfa, and analyzed their effects on viability and pigmentation. Furthermore, we knocked in a fluorescent protein into the mitfa locus, a proof-of-principle experiment of this powerful technology in betta. Finally, we used Tol2-mediated transgenesis to create fish with ubiquitous expression of GFP, and then developed a bicistronic plasmid with heart-specific expression of a red fluorescent protein to serve as a visible marker of successful transgenesis. Our work highlights the potential for the genetic manipulation of betta, providing valuable resources for the effective use of genetic tools in this animal model.

Can the Center for Epidemiologic Studies Depression Scale Be Used to Screen for Depression in Children and Adolescents? An Updated Systematic Review.

This study is an updated systematic review verifying whether the Center for Epidemiologic Studies Depression Scale (CES-D) is a valuable screening tool for children and adolescents. Electronic searches were performed on MEDLINE, EMBASE, CINAHL, and PsycArticles, using depression and CES-D as keywords. Fourteen studies that included 7,843 children and adolescents were analyzed. In the meta-analysis by CES-D type, the pooled sensitivity and specificity for the long version were 0.81 and 0.72, respectively; they were 0.80 and 0.74 for the short version, respectively. The summary receiver operating characteristic (sROC) curves were 0.83 and 0.86, respectively. Compared to the CES-D and other tools, the pooled sensitivity (0.84 vs. 0.83) and the pooled specificity (0.72 vs. 0.74) were similar, and the sROC curve was the same at 0.83. This review indicates that the CES-D is an available and valuable tool for screening depression in children and adolescents.

The evolution of two transmissible cancers in Tasmanian devils.

Tasmanian devils have spawned two transmissible cancer lineages, named devil facial tumor 1 (DFT1) and devil facial tumor 2 (DFT2). We investigated the genetic diversity and evolution of these clones by analyzing 78 DFT1 and 41 DFT2 genomes relative to a newly assembled, chromosome-level reference. Time-resolved phylogenetic trees reveal that DFT1 first emerged in 1986 (1982 to 1989) and DFT2 in 2011 (2009 to 2012). Subclone analysis documents transmission of heterogeneous cell populations. DFT2 has faster mutation rates than DFT1 across all variant classes, including substitutions, indels, rearrangements, transposable element insertions, and copy number alterations, and we identify a hypermutated DFT1 lineage with defective DNA mismatch repair. Several loci show plausible evidence of positive selection in DFT1 or DFT2, including loss of chromosome Y and inactivation of MGA, but none are common to both cancers. This study reveals the parallel long-term evolution of two transmissible cancers inhabiting a common niche in Tasmanian devils.

Silk Hydrogel-Mediated Delivery of Bone Morphogenetic Protein 7 Directly to Subcutaneous White Adipose Tissue Increases Browning and Energy Expenditure.

Objective: Increasing the mass and/or activity of brown adipose tissue (BAT) is one promising avenue for treating obesity and related metabolic conditions, given that BAT has a high potential for energy expenditure and is capable of improving glucose and lipid homeostasis. BAT occurs either in discrete "classical" depots, or interspersed in white adipose tissue (WAT), termed "inducible/recruitable" BAT, or 'beige/brite' adipocytes. We and others have demonstrated that bone morphogenetic protein 7 (BMP7) induces brown adipogenesis in committed and uncommitted progenitor cells, resulting in increased energy expenditure and reduced weight gain in mice. BMP7 is therefore a reliable growth factor to induce browning of WAT. Methods: In this study, we sought to deliver BMP7 specifically to subcutaneous (sc)WAT in order to induce tissue-resident progenitor cells to differentiate into energy-expending recruitable brown adipocytes, without off-target effects like bone formation, which can occur when BMPs are in the presence of bone progenitor cells (outside of WAT). BMP7 delivery directly to WAT may also promote tissue innervation, or directly activate mitochondrial activity in brown adipocytes, as we have demonstrated previously. We utilized silk protein in the form of an injectable hydrogel carrying BMP7. Silk scaffolds are useful for in vivo delivery of substances due to favorable material properties, including controlled release of therapeutic proteins in an active form, biocompatibility with minimal immunogenic response, and prior FDA approval for some medical materials. For this study, the silk was engineered to meet desirable release kinetics for BMP7 in order to mimic our prior in vitro brown adipocyte differentiation studies. Fluorescently-labeled silk hydrogel loaded with BMP7 was directly injected into WAT through the skin and monitored by non-invasive in vivo whole body imaging, including in UCP1-luciferase reporter mice, thereby enabling an approach that is translatable to humans. Results: Injection of the BMP7-loaded silk hydrogels into the subcutaneous WAT of mice resulted in "browning", including the development of multilocular, uncoupling protein 1 (UCP1)-positive brown adipocytes, and an increase in whole-body energy expenditure and skin temperature. In diet-induced obese mice, BMP7-loaded silk delivery to subcutaneous WAT resulted in less weight gain, reduced circulating glucose and lower respiratory exchange ratio (RER). Conclusions: In summary, BMP7 delivery via silk scaffolds directly into scWAT is a novel translational approach to increase browning and energy expenditure, and represents a potential therapeutic avenue for delivering substances directly to adipose depots in pursuit of metabolic treatments.

Care and Use of Siamese Fighting Fish (Betta Splendens) for Research.

Betta splendens, also called Siamese fighting fish or 'betta,' are a popular species in the fishkeeping hobby. Native to South- east Asia, betta have been selectively bred for their fighting ability for hundreds of years, which has resulted in the species' characteristic male aggression. More recently, betta have been bred for a number of ornamental traits such as coloration, fin morphology, and body size. Betta have unique characteristics and an evolutionary history that make them a useful model for studies in the fields of behavior, endocrinology, neurobiology, genetics, development, and evolution. However, standard laboratory procedures for raising and keeping these fish are not well established, which has limited their use. Here, we briefly review the past and present use of betta in research, with a focus on their utility in behavioral, neurobiological, and evolutionary studies. We then describe effective husbandry practices for maintaining betta as a research colony.

Genomic consequences of domestication of the Siamese fighting fish.

Siamese fighting (betta) fish are among the most popular and morphologically diverse pet fish, but the genetic bases of their domestication and phenotypic diversification are largely unknown. We assembled de novo the genome of a wild Betta splendens and whole-genome sequenced 98 individuals across five closely related species. We find evidence of bidirectional hybridization between domesticated ornamental betta and other wild Betta species. We discover dmrt1 as the main sex determination gene in ornamental betta and that it has lower penetrance in wild B. splendens. Furthermore, we find genes with signatures of recent, strong selection that have large effects on color in specific parts of the body or on the shape of individual fins and that most are unlinked. Our results demonstrate how simple genetic architectures paired with anatomical modularity can lead to vast phenotypic diversity generated during animal domestication and launch betta as a powerful new system for evolutionary genetics.

Analysis of a COVID-19 Prescreening Process in an Outpatient Clinic at a University Hospital during the COVID-19 Pandemic.

BACKGROUND: To minimize nosocomial infection against coronavirus disease 2019 (COVID-19), most hospitals conduct a prescreening process to evaluate the patient or guardian of any symptoms suggestive of COVID-19 or exposure to a COVID-19 patient at entrances of hospital buildings. In our hospital, we have implemented a two-level prescreening process in the outpatient clinic: an initial prescreening process at the entrance of the outpatient clinic (PPEO) and a second prescreening process is repeated in each department. If any symptoms or epidemiological history are identified at the second level, an emergency code is announced through the hospital's address system. The patient is then guided outside through a designated aisle. In this study, we analyze the cases missed in the PPEO that caused the emergency code to be applied. METHODS: All cases reported from March 2020 to April 2021 were analyzed retrospectively. We calculated the incidence of cases missed by the PPEO per 1,000 outpatients and compared the incidence between first-time hospital visitors and those visiting for the second time or more; morning and afternoon office hours; and days of the week. RESULTS: During the study period, the emergency code was applied to 449 cases missed by the PPEO. Among those cases, 20.7% were reported in otorhinolaryngology, followed by 11.6% in gastroenterology, 5.8% in urology, and 5.8% in dermatology. Fever was the most common symptom (59.9%), followed by cough (19.8%). The incidence of cases per 1,000 outpatients was significantly higher among first-time visitors than among those visiting for the second time or more (1.77 [confidence interval (CI), 1.44-2.10] vs. 0.59 [CI, 0.52-0.65], respectively) (P < 0.001). CONCLUSION: Fever was the most common symptom missed by the PPEO, and otorhinolaryngology and gastroenterology most frequently reported missed cases. Cases missed by the PPEO were more likely to occur among first-time visitors than returning visitors. The results obtained from this study can provide insights or recommendations to other healthcare facilities in operating prescreening processes during the COVID-19 pandemic.

Surveillance of the infection prevention and control practices of healthcare workers by an infection control surveillance-working group and a team of infection control coordinators during the COVID-19 pandemic.

BACKGROUND: During the ongoing coronavirus disease (COVID-19) pandemic, hospitals have strengthened their guidelines on infection prevention and control (IPC), and a rigorous adherence to these guidelines is crucial. An infection control surveillance-working group (ICS-WG) and infection control coordinators (ICCs) team were created to monitor the IPC practices of the healthcare workers (HCWs) in a regional hospital in Korea. This study analyzed the surveillance results and aimed to identify what IPC practices needed improvement. METHODS: During phase 1 (March to April 2020), the ICS-WG performed random audits, recorded incidences of improper IPC practices, and provided advice to the violators. During phase 2 (April to July), the ICCs inspected the hospital units and proposed practical ideas about IPC. The surveillance and proposals targeted the following practices: patient screening, usage of personal protective equipment (PPE), hand and respiratory hygiene, equipment reprocessing, environmental cleaning, management of medical waste, and social distancing. RESULTS: In phase 1, of the 127 violations observed, most (32.3%) corresponded to hand and respiratory hygiene. In phase 2, the highest proportion of violation per category was observed in the management of medical waste (37.8%); among these, a higher proportion of violation (71.4%) was observed in the collection of medical waste. Of the 106 proposals made by the ICCs, the most addressed practice was patient screening (28.3%). No case of nosocomial infection was reported during the study period. CONCLUSION: Adherence to proper hand and respiratory hygiene was inadequate at the early stage of the COVID-19 pandemic. The results indicate that more attention and further training are needed for the management of medical waste, particularly medical waste collection, and that continuous upgrading of the strategies for patient screening is essential. These results will be useful in helping other healthcare facilities to establish their IPC strategies.

Satisfaction Survey of Patients and Medical Staff for Telephone-Based Telemedicine During Hospital Closing Due to COVID-19 Transmission.

Introduction: Telephone-based telemedicine was temporarily permitted in Korea during the COVID-19 pandemic. The purpose of this study was to assess satisfaction with the telemedicine done during temporary hospital closing when in-person visits were not allowed due to in-hospital COVID-19 transmission. Methods: Survey questionnaires partially taken from a telehealth usability questionnaire (TUQ) were sent to 6,840 patients who used telephone-based telemedicine from February 24 to March 7, 2020. Questionnaires sent to patients and additionally created questionnaires to evaluate telemedicine were sent to medical staff (182 doctors and 138 nurses). Results: Response rates of patients and medical staff were 13.2% and 17.2%, respectively. Patients' satisfaction with telemedicine was significantly greater than medical staff's satisfaction for all five components taken from TUQ (all p = 0.000). In addition, created questionnaires showed good reliability, obtaining similar results between doctors and nurses (all p > 0.05). More than 85% of medical staff replied that telemedicine was needed in COVID-19, whereas more than 80% of them worried about incomplete assessment and communication of medical condition. Overall satisfaction with telemedicine by medical staff was 49.7%. The strength of telephone-based telemedicine was patients' convenience (53.4%). However, incomplete assessment of patients' condition (55.0%) was its weakness. Conclusion: Satisfaction with telephone-based telemedicine by patients was significantly greater than that by medical staff (doctors and nurses). Negative views for safety and inconvenience resulted in a greater proportion of dissatisfaction among medical staff. For safe application of telemedicine, medical staff insisted that developing a platform and creating guidelines should be needed.

Evolution and lineage dynamics of a transmissible cancer in Tasmanian devils.

Devil facial tumour 1 (DFT1) is a transmissible cancer clone endangering the Tasmanian devil. The expansion of DFT1 across Tasmania has been documented, but little is known of its evolutionary history. We analysed genomes of 648 DFT1 tumours collected throughout the disease range between 2003 and 2018. DFT1 diverged early into five clades, three spreading widely and two failing to persist. One clade has replaced others at several sites, and rates of DFT1 coinfection are high. DFT1 gradually accumulates copy number variants (CNVs), and its telomere lengths are short but constant. Recurrent CNVs reveal genes under positive selection, sites of genome instability, and repeated loss of a small derived chromosome. Cultured DFT1 cell lines have increased CNV frequency and undergo highly reproducible convergent evolution. Overall, DFT1 is a remarkably stable lineage whose genome illustrates how cancer cells adapt to diverse environments and persist in a parasitic niche.

Generation, Coordination, and Evolution of Neural Circuits for Vocal Communication.

In many species, vocal communication is essential for coordinating social behaviors including courtship, mating, parenting, rivalry, and alarm signaling. Effective communication requires accurate production, detection, and classification of signals, as well as selection of socially appropriate responses. Understanding how signals are generated and how acoustic signals are perceived is key to understanding the neurobiology of social behaviors. Here we review our long-standing research program focused on Xenopus, a frog genus which has provided valuable insights into the mechanisms and evolution of vertebrate social behaviors. In Xenopus laevis, vocal signals differ between the sexes, through development, and across the genus, reflecting evolutionary divergence in sensory and motor circuits that can be interrogated mechanistically. Using two ex vivo preparations, the isolated brain and vocal organ, we have identified essential components of the vocal production system: the sexually differentiated larynx at the periphery, and the hindbrain vocal central pattern generator (CPG) centrally, that produce sex- and species-characteristic sound pulse frequencies and temporal patterns, respectively. Within the hindbrain, we have described how intrinsic membrane properties of neurons in the vocal CPG generate species-specific vocal patterns, how vocal nuclei are connected to generate vocal patterns, as well as the roles of neurotransmitters and neuromodulators in activating the circuit. For sensorimotor integration, we identified a key forebrain node that links auditory and vocal production circuits to match socially appropriate vocal responses to acoustic features of male and female calls. The availability of a well supported phylogeny as well as reference genomes from several species now support analysis of the genetic architecture and the evolutionary divergence of neural circuits for vocal communication. Xenopus thus provides a vertebrate model in which to study vocal communication at many levels, from physiology, to behavior, and from development to evolution. As one of the most comprehensively studied phylogenetic groups within vertebrate vocal communication systems, Xenopus provides insights that can inform social communication across phyla.

Tracing the rise of malignant cell lines: Distribution, epidemiology and evolutionary interactions of two transmissible cancers in Tasmanian devils.

Emerging infectious diseases are rising globally and understanding host-pathogen interactions during the initial stages of disease emergence is essential for assessing potential evolutionary dynamics and designing novel management strategies. Tasmanian devils (Sarcophilus harrisii) are endangered due to a transmissible cancer-devil facial tumour disease (DFTD)-that since its emergence in the 1990s, has affected most populations throughout Tasmania. Recent studies suggest that devils are adapting to the DFTD epidemic and that disease-induced extinction is unlikely. However, in 2014, a second and independently evolved transmissible cancer-devil facial tumour 2 (DFT2)-was discovered at the d'Entrecasteaux peninsula, in south-east Tasmania, suggesting that the species is prone to transmissible cancers. To date, there is little information about the distribution, epidemiology and effects of DFT2 and its interaction with DFTD. Here, we use data from monitoring surveys and roadkills found within and adjacent to the d'Entrecasteaux peninsula to determine the distribution of both cancers and to compare their epidemiological patterns. Since 2012, a total of 51 DFTD tumours have been confirmed among 26 individuals inside the peninsula and its surroundings, while 40 DFT2 tumours have been confirmed among 23 individuals, and two individuals co-infected with both tumours. All devils with DFT2 were found within the d'Entrecasteaux peninsula, suggesting that this new transmissible cancer is geographically confined to this area. We found significant differences in tumour bodily location in DFTD and DFT2, with non-facial tumours more commonly found in DFT2. There was a significant sex bias in DFT2, with most cases reported in males, suggesting that since DFT2 originated from a male host, females might be less susceptible to this cancer. We discuss the implications of our results for understanding the epidemiological and evolutionary interactions of these two contemporary transmissible cancers and evaluating the effectiveness of potential management strategies.

Somatic evolution and global expansion of an ancient transmissible cancer lineage.

The canine transmissible venereal tumor (CTVT) is a cancer lineage that arose several millennia ago and survives by "metastasizing" between hosts through cell transfer. The somatic mutations in this cancer record its phylogeography and evolutionary history. We constructed a time-resolved phylogeny from 546 CTVT exomes and describe the lineage's worldwide expansion. Examining variation in mutational exposure, we identify a highly context-specific mutational process that operated early in the cancer's evolution but subsequently vanished, correlate ultraviolet-light mutagenesis with tumor latitude, and describe tumors with heritable hyperactivity of an endogenous mutational process. CTVT displays little evidence of ongoing positive selection, and negative selection is detectable only in essential genes. We illustrate how long-lived clonal organisms capture changing mutagenic environments, and reveal that neutral genetic drift is the dominant feature of long-term cancer evolution.

Predicting Response to Neoadjuvant Chemotherapy in Patients With Breast Cancer: Combined Statistical Modeling Using Clinicopathological Factors and FDG PET/CT Texture Parameters.

PURPOSE: The aim of this study was to develop a combined statistical model using both clinicopathological factors and texture parameters from F-FDG PET/CT to predict responses to neoadjuvant chemotherapy in patients with breast cancer. MATERIALS AND METHODS: A total of 435 patients with breast cancer were retrospectively enrolled. Clinical and pathological data were obtained from electronic medical records. Texture parameters were extracted from pretreatment FDG PET/CT images. The end point was pathological complete response, defined as the absence of residual disease or the presence of residual ductal carcinoma in situ without residual lymph node metastasis. Multivariable logistic regression modeling was performed using clinicopathological factors and texture parameters as covariates. RESULTS: In the multivariable logistic regression model, various factors and parameters, including HER2, histological grade or Ki-67, gradient skewness, gradient kurtosis, contrast, difference variance, angular second moment, and inverse difference moment, were selected as significant prognostic variables. The predictive power of the multivariable logistic regression model incorporating both clinicopathological factors and texture parameters was significantly higher than that of a model with only clinicopathological factors (P = 0.0067). In subgroup analysis, texture parameters, including gradient skewness and gradient kurtosis, were selected as independent prognostic factors in the HER2-negative group. CONCLUSIONS: A combined statistical model was successfully generated using both clinicopathological factors and texture parameters to predict the response to neoadjuvant chemotherapy. Results suggest that addition of texture parameters from FDG PET/CT can provide more information regarding treatment response prediction compared with clinicopathological factors alone.

Tasman-PCR: a genetic diagnostic assay for Tasmanian devil facial tumour diseases.

Tasmanian devils have spawned two transmissible cancer clones, known as devil facial tumour 1 (DFT1) and devil facial tumour 2 (DFT2). DFT1 and DFT2 are transmitted between animals by the transfer of allogeneic contagious cancer cells by biting, and both cause facial tumours. DFT1 and DFT2 tumours are grossly indistinguishable, but can be differentiated using histopathology, cytogenetics or genotyping of polymorphic markers. However, standard diagnostic methods require specialist skills and equipment and entail long processing times. Here, we describe Tasman-PCR: a simple polymerase chain reaction (PCR)-based diagnostic assay that identifies and distinguishes DFT1 and DFT2 by amplification of DNA spanning tumour-specific interchromosomal translocations. We demonstrate the high sensitivity and specificity of this assay by testing DNA from 546 tumours and 804 normal devils. A temporal-spatial screen confirmed the reported geographic ranges of DFT1 and DFT2 and did not provide evidence of additional DFT clones. DFT2 affects disproportionately more males than females, and devils can be co-infected with DFT1 and DFT2. Overall, we present a PCR-based assay that delivers rapid, accurate and high-throughput diagnosis of DFT1 and DFT2. This tool provides an additional resource for devil disease management and may assist with ongoing conservation efforts.

The newly-arisen Devil facial tumour disease 2 (DFT2) reveals a mechanism for the emergence of a contagious cancer.

Devil Facial Tumour 2 (DFT2) is a recently discovered contagious cancer circulating in the Tasmanian devil (Sarcophilus harrisii), a species which already harbours a more widespread contagious cancer, Devil Facial Tumour 1 (DFT1). Here we show that in contrast to DFT1, DFT2 cells express major histocompatibility complex (MHC) class I molecules, demonstrating that loss of MHC is not necessary for the emergence of a contagious cancer. However, the most highly expressed MHC class I alleles in DFT2 cells are common among host devils or non-polymorphic, reducing immunogenicity in a population sharing these alleles. In parallel, MHC class I loss is emerging in vivo, thus DFT2 may be mimicking the evolutionary trajectory of DFT1. Based on these results we propose that contagious cancers may exploit partial histocompatibility between the tumour and host, but that loss of allogeneic antigens could facilitate widespread transmission of DFT2.

The evolutionary history of dogs in the Americas.

Dogs were present in the Americas before the arrival of European colonists, but the origin and fate of these precontact dogs are largely unknown. We sequenced 71 mitochondrial and 7 nuclear genomes from ancient North American and Siberian dogs from time frames spanning ~9000 years. Our analysis indicates that American dogs were not derived from North American wolves. Instead, American dogs form a monophyletic lineage that likely originated in Siberia and dispersed into the Americas alongside people. After the arrival of Europeans, native American dogs almost completely disappeared, leaving a minimal genetic legacy in modern dog populations. The closest detectable extant lineage to precontact American dogs is the canine transmissible venereal tumor, a contagious cancer clone derived from an individual dog that lived up to 8000 years ago.

The Origins and Vulnerabilities of Two Transmissible Cancers in Tasmanian Devils.

Transmissible cancers are clonal lineages that spread through populations via contagious cancer cells. Although rare in nature, two facial tumor clones affect Tasmanian devils. Here we perform comparative genetic and functional characterization of these lineages. The two cancers have similar patterns of mutation and show no evidence of exposure to exogenous mutagens or viruses. Genes encoding PDGF receptors have copy number gains and are present on extrachromosomal double minutes. Drug screening indicates causative roles for receptor tyrosine kinases and sensitivity to inhibitors of DNA repair. Y chromosome loss from a male clone infecting a female host suggests immunoediting. These results imply that Tasmanian devils may have inherent susceptibility to transmissible cancers and present a suite of therapeutic compounds for use in conservation.

The genetic basis of parental care evolution in monogamous mice.

Parental care is essential for the survival of mammals, yet the mechanisms underlying its evolution remain largely unknown. Here we show that two sister species of mice, Peromyscus polionotus and Peromyscus maniculatus, have large and heritable differences in parental behaviour. Using quantitative genetics, we identify 12 genomic regions that affect parental care, 8 of which have sex-specific effects, suggesting that parental care can evolve independently in males and females. Furthermore, some regions affect parental care broadly, whereas others affect specific behaviours, such as nest building. Of the genes linked to differences in nest-building behaviour, vasopressin is differentially expressed in the hypothalamus of the two species, with increased levels associated with less nest building. Using pharmacology in Peromyscus and chemogenetics in Mus, we show that vasopressin inhibits nest building but not other parental behaviours. Together, our results indicate that variation in an ancient neuropeptide contributes to interspecific differences in parental care.

Predictive Validity of Pressure Ulcer Risk Assessment Tools for Elderly: A Meta-Analysis.

Preventing pressure ulcers is one of the most challenging goals existing for today's health care provider. Currently used tools which assess risk of pressure ulcer development rarely evaluate the accuracy of predictability, especially in older adults. The current study aimed at providing a systemic review and meta-analysis of 29 studies using three pressure ulcer risk assessment tools: Braden, Norton, and Waterlow Scales. Overall predictive validities of pressure ulcer risks in the pooled sensitivity and specificity indicated a similar range with a moderate accuracy level in all three scales, while heterogeneity showed more than 80% variability among studies. The studies applying the Braden Scale used five different cut-off points representing the primary cause of heterogeneity. Results indicate that commonly used screening tools for pressure ulcer risk have limitations regarding validity and accuracy for use with older adults due to heterogeneity among studies.