The pRb/RBL2-E2F1/4-GCN5 axis regulates cancer stem cell formation and G0 phase entry/exit by paracrine mechanisms.

The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.

Molecular identification of Crimean-Congo haemorrhagic fever virus in Hyalomma rufipes and Amblyomma variegatum in the Upper East Region of Ghana.

Sampled ticks were screened for Crimean-Congo haemorrhagic fever virus (CCHFV) using an assay that targets the nucleoprotein gene region of the S segment, a conserved region of the CCHFV genome. Minimum infection rates of 0.34% and 0.10% were obtained when testing pools of Hyalomma rufipes and Amblyomma variegatum, respectively. Next-generation sequencing and phylogenetic analysis showed that the S and L segments of the CCHFV isolate clustered with those of similar isolates of genotype III. However, analysis of the M segment showed that reassortment had occurred, causing this segment to cluster with those of isolates of genotype I, providing the first evidence of such an occurrence in Ghana.

Spatial and Seasonal Patterns of Tick Infestations in Kassena-Nankana Livestock.

The ability of ticks to adapt to different ecological zones, coupled with the spread of infectious pathogens negatively affects livestock production and thus, there is a need for better control strategies. However, control measures within a geographical region can only be effective if there is available information on tick population dynamics and ecology. This study focused on ticks infesting livestock in the Kassena-Nankana Districts of the Upper East Region of Ghana. The ticks were morphologically identified, variables such as season, animal host, and predilection sites were recorded, and the data were analyzed using STATA version 13. Out of 448 livestock examined, tick infestation in cattle was (78.60%), followed by sheep (25%) and goats (5.88%). A total of 1,550 ticks including nymphs (303) and adults (1,247) were collected. Adult ticks were found to be significantly associated with season (p < 0.001), with a high burden in the wet season. The nymph burden and body parts of livestock hosts were significantly associated with more nymphs collected from male animals than females (p < 0.001). Three genera of ticks, Amblyomma (62.97%), Hyalomma (18.71%), and Rhipicephalus (18.32%) were morphologically identified with the most predominant tick species recorded as Amblyomma variegatum (62.97%). Matured A. variegatum was sampled primarily in the wet season with their predilection site as the udder/scrotum (p < 0.001). However, adult Hyalomma truncatum was observed to have a significant association with the anal region (p < 0.001). Findings from this study are essential for formulating tick control measures to prevent the spread of infectious pathogens.

BRD9-SMAD2/3 Orchestrates Stemness and Tumorigenesis in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. The epigenetic mechanisms regulating CSCs are currently insufficiently understood, which hampers the development of novel strategies for eliminating CSCs. METHODS: By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodeling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signaling pathway. RESULTS: Inhibition and genetic ablation of BRD9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumors from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. CONCLUSIONS: Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases.

BACKGROUND: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. METHODS: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. RESULTS: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. CONCLUSIONS: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.

First record of Babesia and Theileria parasites in ticks from Kassena-Nankana, Ghana.

Ticks are efficient vectors for transmitting pathogens that negatively affect livestock production and pose a risk to public health. In this study, Babesia and Theileria species were identified in ticks collected from cattle, sheep and goats from the Kassena-Nankana Districts of Ghana between February and December 2020. A total of 1550 ticks were collected, morphologically identified, pooled and screened for pathogens using primers that amplify a 560 bp fragment of the ssrRNA gene and Sanger sequencing. Amblyomma variegatum (62.98%) was the predominant tick species. From the 491 tick pools screened, 12/15 (2.44%) positive pools were successfully sequenced. The pathogen DNA identified were Theileria ovis in eight (15.38%) pools of Rhipicephalus evertsi evertsi, Theileria velifera in two (0.78%) pools of A. variegatum and Babesia occultans and Babesia sp. Xinjiang in one (1.72%) pool each of Hyalomma truncatum. It was further observed that T. ovis occurred in ticks collected from only sheep (p < 0.001) which were females (p = 0.023) and < =1 year old (p = 0.040). This study reports the first identification of these pathogens in ticks within Kassena-Nankana. With the constant trade of livestock, there is a need for effective tick control measures to prevent infection spread.

A geopositioned and evidence-graded pan-species compendium of Mayaro virus occurrence.

Mayaro Virus (MAYV) is an emerging health threat in the Americas that can cause febrile illness as well as debilitating arthralgia or arthritis. To better understand the geographic distribution of MAYV risk, we developed a georeferenced database of MAYV occurrence based on peer-reviewed literature and unpublished reports. Here we present this compendium, which includes both point and polygon locations linked to occurrence data documented from its discovery in 1954 until 2022. We describe all methods used to develop the database including data collection, georeferencing, management and quality-control. We also describe a customized grading system used to assess the quality of each study included in our review. The result is a comprehensive, evidence-graded database of confirmed MAYV occurrence in humans, non-human animals, and arthropods to-date, containing 262 geo-positioned occurrences in total. This database - which can be updated over time - may be useful for local spill-over risk assessment, epidemiological modelling to understand key transmission dynamics and drivers of MAYV spread, as well as identification of major surveillance gaps.

Occurrence of Rickettsia spp. and Coxiella burnetii in ixodid ticks in Kassena-Nankana, Ghana.

Ticks are arthropods of veterinary and medical importance which spread zoonotic pathogens that link animal and human health. In this study, ticks were collected from 448 livestock between February and December 2020 in the Kassena-Nankana Districts of Ghana and screened for the presence of zoonotic pathogens DNA using PCR and sequencing approaches. In total, 1550 ticks were collected and morphologically identified. Three tick genera were identified with Amblyomma variegatum (63%) as the predominant tick species collected. DNA was extracted from 491 tick pools and screened for the presence of DNA of Rickettsia spp. based on the 115 bp fragment of the 17 kDa surface protein and 639 bp of the Outer membrane protein A (ompA) gene and the 295 bp fragment of the transposase gene of Coxiella burnetii IS1111a element. From the 491 pools screened, the DNA of Rickettsia spp. and C. burnetii was detected in 56.8 and 3.7%, respectively. Coinfections were identified in 2.4% of the tick pools. Characterization of the Rickettsia spp. in this study based on the ompA gene showed that the DNA of Rickettsia africae and Rickettsia aeschlimannii accounted for 39.7 and 14.7%, respectively, and were 100% similar to sequences in GenBank. Most R. africae and C. burnetii infections occurred in ticks collected in the wet season, whereas R. aeschlimannii occurred mostly in the dry season. These pathogens are potential public health threats, thus there is a need to implement control measures to reduce the risk of infections in vulnerable populations.

HSD3B1 is an oxysterol 3ß-hydroxysteroid dehydrogenase in human placenta.

Most biologically active oxysterols have a 3ß-hydroxy-5-ene function in the ring system with an additional site of oxidation at C-7 or on the side-chain. In blood plasma oxysterols with a 7α-hydroxy group are also observed with the alternative 3-oxo-4-ene function in the ring system formed by ubiquitously expressed 3ß-hydroxy-Δ5-C27-steroid oxidoreductase Δ5-isomerase, HSD3B7. However, oxysterols without a 7α-hydroxy group are not substrates for HSD3B7 and are not usually observed with the 3-oxo-4-ene function. Here we report the unexpected identification of oxysterols in plasma derived from umbilical cord blood and blood from pregnant women taken before delivery at 37+ weeks of gestation, of side-chain oxysterols with a 3-oxo-4-ene function but no 7α-hydroxy group. These 3-oxo-4-ene oxysterols were also identified in placenta, leading to the hypothesis that they may be formed by a previously unrecognized 3ß-hydroxy-Δ5-C27-steroid oxidoreductase Δ5-isomerase activity of HSD3B1, an enzyme which is highly expressed in placenta. Proof-of-principle experiments confirmed that HSD3B1 has this activity. We speculate that HSD3B1 in placenta is the source of the unexpected 3-oxo-4-ene oxysterols in cord and pregnant women's plasma and may have a role in controlling the abundance of biologically active oxysterols delivered to the fetus.

Discovery of a Potent and Selective Naphthyridine-Based Chemical Probe for Casein Kinase 2.

Naphthyridine-based inhibitors were synthesized to yield a potent and cell-active inhibitor of casein kinase 2 (CK2). Compound 2 selectively inhibits CK2α and CK2α' when profiled broadly, thereby making it an exquisitely selective chemical probe for CK2. A negative control that is structurally related but lacks a key hinge-binding nitrogen (7) was designed on the basis of structural studies. Compound 7 does not bind CK2α or CK2α' in cells and demonstrates excellent kinome-wide selectivity. Differential anticancer activity was observed when compound 2 was profiled alongside a structurally distinct CK2 chemical probe: SGC-CK2-1. This naphthyridine-based chemical probe (2) represents one of the best available small molecule tools with which to interrogate biology mediated by CK2.

BRD9-SMAD2/3 orchestrates stemness and tumorigenesis in pancreatic ductal adenocarcinoma.

The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodelling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signalling pathway. Inhibition and genetic ablation of BDR9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumours from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

Molecular survey of Anaplasma and Ehrlichia species in livestock ticks from Kassena-Nankana, Ghana; with a first report of Anaplasma capra and Ehrlichia minasensis.

Tick-borne pathogens harm livestock production and pose a significant risk to public health. To combat these effects, it is necessary to identify the circulating pathogens to create effective control measures. This study identified Anaplasma and Ehrlichia species in ticks collected from livestock in the Kassena-Nankana Districts between February 2020 and December 2020. A total of 1550 ticks were collected from cattle, sheep and goats. The ticks were morphologically identified, pooled and screened for pathogens using primers that amplify a 345 bp fragment of the 16SrRNA gene and Sanger sequencing. The predominant tick species collected was Amblyomma variegatum (62.98%). From the 491 tick pools screened, 34 (6.92%) were positive for Ehrlichia and Anaplasma. The pathogens identified were Ehrlichia canis (4.28%), Ehrlichia minasensis (1.63%), Anaplasma capra (0.81%) and Anaplasma marginale (0.20%). This study reports the first molecular identification of the above-mentioned Ehrlichia and Anaplasma species in ticks from Ghana. With the association of human infections with the zoonotic pathogen A. capra, livestock owners are at risk of infections, calling for the development of effective control measures.

Prolyl-tRNA synthetase as a novel therapeutic target in multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.

Assessment of Dermal and Inhalation Exposure to Permethrin During Field Treatment of Military Uniforms.

INTRODUCTION: Permethrin is a common pesticide spray-applied to civilian clothing and military uniforms for protection against biting arthropods in an effort to reduce risks to arthropod-borne diseases. During mass clothing spray events, exposure is possible through the dermal, inhalation, and ingestion routes. The potentially exposed population during a spray event includes the pesticide applicator(s) and working party (personnel who handle clothing/uniforms by positioning on the ground, flipping, and removing after spraying is complete). Previous investigation is limited regarding permethrin exposure via multiple routes of entry. Additionally, most exposure assessments are limited to pesticide applicators rather than working party that support applicator personnel. The purpose of this investigation was to conduct a multi-route exposure assessment for all personnel normally participating in mass permethrin military uniform treatments. MATERIALS AND METHODS: The protocol and Informed Consent Document were approved by the Uniformed Services University of the Health Sciences (USUHS) Institutional Review Board (IRB) before fieldwork initiation (IRB number: USUHS.2019-032). Sampling occurred during routine spray events performed by 14 U.S. Navy personnel (3 applicators and 11 working party) over 2 days. Personal exposures were measured with dermal sampling, and airborne concentrations were measured with area air sampling. Permethrin area air sampling and analysis were conducted using OSHA Versatile Sampler-2 sampling media (n = 36). Dermal exposure was measured using dosimeter gloves (n = 26) and a dermal patch (n = 26) worn by study participants. RESULTS: All air samples were reported below the 0.4 µg limit of quantification. Glove sample results ranged from 45 to 120,000 µg and patch results ranged from 0.57 to 45 µg. A repeated-measures ANOVA showed non-statistically significant differences in dermal concentrations (P-value = .8340) between the applicators and working party in patches and gloves. CONCLUSIONS: Results suggest dermal contact is the primary route of exposure compared to inhalation when mass spraying clothing with permethrin. Similar dermal exposures between these two occupations may necessitate reconsidering risk assessment procedures, training and personal protective equipment (PPE) requirements for mass spray uniform treatments. Specifically, while PPE requirements for applicators are highly regulated by the Armed Forces Pest Management Board and include items such as Tyvek suits, chemical protective gloves, and air-purifying respirators, PPE was not regulated for working party personnel before this investigation.

Minimal Rickettsial Infection Rates and Distribution of Ticks in Uganda: An Assessment of the Seasonal Effects and Relevance to Tick-Borne Disease Risk in East Africa.

Tick-borne diseases (TBDs) pose a significant risk to humans and represent one of the major factors influencing readiness within the United States' military worldwide. Additionally, ticks and TBDs constitute major animal health problems leading to economic losses at multiple levels affecting low- and middle-income countries the hardest. Tick control is frequently hampered by issues ranging from acaricide resistance to lack of data on tick distribution and infection rates. We conducted a cross-sectional study to assess tick species distribution, host use, and rickettsial pathogen infection rate of ticks in different areas of the Uganda Cattle Corridor. We identified 4,425 hard ticks (Ixodida: Ixodidae) comprised of seven species by morphological characters with 3,315 ticks collected from four locations during the dry season and 1,110 ticks from one location during the wet season. Rickettsial pathogen prevalence was assessed in ticks collected from two districts to determine the minimum infection rate compared across seasons, village location, and tick species. We found statistically significant differences in the abundance and distribution of tick species among districts in the dry season, host animal species, and the proportion of rickettsial positive pools between villages. Seasonality, village location, and tick species do not affect the minimum infection rate of rickettsial pathogens of ticks in Uganda, but village location affects the proportion of positive tick pools. These results indicate geographical and seasonal differences among pathogen-harboring ticks contributing to our understanding of the current distribution of ticks and TBDs in Uganda.

BRD9-containing non-canonical BAF complex maintains somatic cell transcriptome and acts as a barrier to human reprogramming.

Epigenetic reprogramming to pluripotency requires extensive remodeling of chromatin landscapes to silence existing cell-type-specific genes and activate pluripotency genes. ATP-dependent chromatin remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of recently identified Bromodomain-containing protein 9 (BRD9) and the associated non-canonical BRG1-associated factors (ncBAF) complex in reprogramming remains unknown. Here, we show that genetic or chemical inhibition of BRD9, as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase human somatic cell reprogramming efficiency and can replace KLF4 and c-MYC. We find that BRD9 is dispensable for human induced pluripotent stem cells under primed but not under naive conditions. Mechanistically, BRD9 inhibition downregulates fibroblast-related genes and decreases chromatin accessibility at somatic enhancers. BRD9 maintains the expression of transcriptional regulators MN1 and ZBTB38, both of which impede reprogramming. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming.

An ecological niche model to predict the geographic distribution of Haemagogus janthinomys, Dyar, 1921 a yellow fever and Mayaro virus vector, in South America.

Yellow fever virus (YFV) has a long history of impacting human health in South America. Mayaro virus (MAYV) is an emerging arbovirus of public health concern in the Neotropics and its full impact is yet unknown. Both YFV and MAYV are primarily maintained via a sylvatic transmission cycle but can be opportunistically transmitted to humans by the bites of infected forest dwelling Haemagogus janthinomys Dyar, 1921. To better understand the potential risk of YFV and MAYV transmission to humans, a more detailed understanding of this vector species' distribution is critical. This study compiled a comprehensive database of 177 unique Hg. janthinomys collection sites retrieved from the published literature, digitized museum specimens and publicly accessible mosquito surveillance data. Covariate analysis was performed to optimize a selection of environmental (topographic and bioclimatic) variables associated with predicting habitat suitability, and species distributions modelled across South America using a maximum entropy (MaxEnt) approach. Our results indicate that suitable habitat for Hg. janthinomys can be found across forested regions of South America including the Atlantic forests and interior Amazon.

Bisphosphonates: The role of chemistry in understanding their biological actions and structure-activity relationships, and new directions for their therapeutic use.

The bisphosphonates ((HO)2P(O)CR1R2P(O)(OH)2, BPs) were first shown to inhibit bone resorption in the 1960s, but it was not until 30 years later that a detailed molecular understanding of the relationship between their varied chemical structures and biological activity was elucidated. In the 1990s and 2000s, several potent bisphosphonates containing nitrogen in their R2 side chains (N-BPs) were approved for clinical use including alendronate, risedronate, ibandronate, and zoledronate. These are now mostly generic drugs and remain the leading therapies for several major bone-related diseases, including osteoporosis and skeletal-related events associated with bone metastases. The early development of chemistry in this area was largely empirical and only a few common structural features related to strong binding to calcium phosphate were clear. Attempts to further develop structure-activity relationships to explain more dramatic pharmacological differences in vivo at first appeared inconclusive, and evidence for mechanisms underlying cellular effects on osteoclasts and macrophages only emerged after many years of research. The breakthrough came when the intracellular actions on the osteoclast were first shown for the simpler bisphosphonates, via the in vivo formation of P-C-P derivatives of ATP. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates in the 1980s and 1990s led to the key discovery that the antiresorptive effects of these more complex analogs on osteoclasts result mostly from their potency as inhibitors of the enzyme farnesyl diphosphate synthase (FDPS/FPPS). This key branch-point enzyme in the mevalonate pathway of cholesterol biosynthesis is important for the generation of isoprenoid lipids that are utilized for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Since then, it has become even more clear that the overall pharmacological effects of individual bisphosphonates on bone depend upon two key properties: the affinity for bone mineral and inhibitory effects on biochemical targets within bone cells, in particular FDPS. Detailed enzyme-ligand crystal structure analysis began in the early 2000s and advances in our understanding of the structure-activity relationships, based on interactions with this target within the mevalonate pathway and related enzymes in osteoclasts and other cells have continued to be the focus of research efforts to this day. In addition, while many members of the bisphosphonate drug class share common properties, now it is more clear that chemical modifications to create variations in these properties may allow customization of BPs for different uses. Thus, as the appreciation for new potential opportunities with this drug class grows, new chemistry to allow ready access to an ever-widening variety of bisphosphonates continues to be developed. Potential new uses of the calcium phosphate binding mechanism of bisphosphonates for the targeting of other drugs to the skeleton, and effects discovered on other cellular targets, even at non-skeletal sites, continue to intrigue scientists in this research field.