For the last 40 years, the study of cooperative breeding (CB) in birds has proceeded primarily in the context of discovering the ecological, geographical, and behavioral drivers of helping. The advent of molecular tools in the early 1990s assisted in clarifying the relatedness of helpers to those helped, in some cases, confirming predictions of kin selection theory. Methods for genome-wide analysis of sequence variation, gene expression, and epigenetics promise to add new dimensions to our understanding of avian CB, primarily in the area of molecular and developmental correlates of delayed breeding and dispersal, as well as the ontogeny of achieving parental status in nature. Here, we outline key ways in which modern -omics approaches, in particular genome sequencing, transcriptomics, and epigenetic profiling such as ATAC-seq, can be used to add a new level of analysis of avian CB. Building on recent and ongoing studies of avian social behavior and sociogenomics, we review how high-throughput sequencing of a focal species or clade can provide a robust foundation for downstream, context-dependent destructive and non-destructive sampling of specific tissues or physiological states in the field for analysis of gene expression and epigenetics. -Omics approaches have the potential to inform not only studies of the diversification of CB over evolutionary time, but real-time analyses of behavioral interactions in the field or lab. Sociogenomics of birds represents a new branch in the network of methods used to study CB, and can help clarify ways in which the different levels of analysis of CB ultimately interact in novel and unexpected ways. Cooperative breeding (CB) in birds was first described in detail by tropical naturalist Alexander Skutch in 1935. He observed extra-parental behavior, which he defined as non-parental birds assisting breeding by bringing food to fledglings and females in the nest . Darwin Following Building on early explorations of the comparative method , second-Although the ecological function and evolutionary origin of CB have been explored for decades with varying degrees of success , TinbergIn the same way that ecological studies identify potential behavioral targets of investigation with -omics methods; -omics methods can greatly clarify potential constraints that could mold the expression of CB and its ability to change when the physical or social environment changes . For exaCB can be considered an emergent property emanating from the interaction of individual behaviors, such as delayed dispersal, territoriality, kin recognition, and parental care . We explResearch with both natural and experimental populations of birds has helped identify consistent behavioral, demographic, and ontogenetic features of individuals in CB systems that in turn provide targets for genomic interrogation. Individual behaviors, such as delayed dispersal, territoriality, kin recognition, and parental care, are intrinsic to CB, which thus can be considered an emergent property from the interaction of these individual behaviors  and 2.One essential characteristic of CB is the presence of delayed dispersal of juveniles from a social unit . DispersMesitornis variegate; Delayed dispersal is essential for the development of CB behavior, and is crucial to the formation of families and groups . In someOne recent argument holds that dispersal and social behavior co-evolve, producing two social morphs: a sessile morph which increases the reproduction of relatives from a specific social group and a dispersive morph which increases its own reproduction . SimulatTerritoriality merits special attention due to two key facts. First, territoriality can influence dispersal . DefendiAnother example of an individual behavior facilitating CB is parental and conspecific recognition, which has been hypothesized to require an identifiable phenotype , as wellAegithalos caudatus use learned vocal signals for kin recognition  use odor to recognize related conspecifics or family members  is a set of immune genes with roles in disease resistance , kin recAlmost all bird species provide some type of parental care, including female uniparental care (8% of avian species), male uniparental care (1% of avian species), CB with more than 2 related individuals caring for young in the same nest (9% of avian species), and biparental care  is higher in nesting birds in both sexes , for which there is an extensive long-term pedigree and blood samples suitable for genetic analysis  species from the wild are moved to the lab to isolate environmental effects and distinguish them from genetic effects ; (2) expThe next best strategy to integrate behaviors in the wild with an -omics approach is behavioral studies in the laboratory. Such studies have been used extensively to understand mechanisms of behavior in vertebrates, such as parental care , aggressComparative genomics offers an important avenue for understanding the genetic basis of CB in birds. Most examples of the genetic basis of social and parental behavior in animals have been discovered at least in part through laboratory crosses, experimental manipulations, or genetic characterization and comparison of inbred strains . Some orComparative analyses of discrete and continuous traits on phylogenetic trees have become quite sophisticated in recent years, with diverse models describing how traits evolve on trees, how natural selection on traits can be detected, and how changes in traits, such as behavior, can be associated with changes in multiple variables . Such anAs recognized long ago by the founders of comparative methods , multiplFor these reasons, convergent evolution has become a key focus in the effort to link genomic change with phenotypic traits in animals . In the A variety of recent examples from birds exploit convergent evolution to understand the origins of complex phenotypic traits, such as loss of flight , vocal lSeveral phylogenetic and statistical approaches can facilitate our understanding of the genomic sources of CB, especially under a scenario of convergent evolution . CompariAdditionally, efforts to measure gene expression differences between species or life history stages are very sensitive to a variety of pitfalls, including presence of paralogs, biases in mapping to reference genomes, and other complications such as incorrect estimation of the effective length of transcripts and genes . Moreovede novo in different clades, suggesting the origin of new genic and regulatory functions not found in other clades , and assays for Transposase-Accessible Chromatin-using sequencing (ATAC-seq) , it is preceptor . In socireceptor . In mammreceptor  TogetherAphelocoma or Cyanocorax and developing longitudinal studies of ontogeny and collecting samples in a field setting from each age-related phenotype from CB and non-CB species, complemented with transcriptomes of blood, brain, and gonads and species-specific annotated reference genomes. A comparative transcriptomic analysis will be conducted and expanded to a comparative phylogenomic analysis to answer questions about how CB has evolved in these clades.In our current work we are studying New World jays such as Distinguishing different forms of CB in birds and the homologies between them is a prerequisite for developing hypotheses for the molecular basis of CB, especially in a comparative framework , 1994. NDue to the plasticity and dependence of diverse fundamental brain and behavioral processes, studying the molecular basis of a complex trait such as CB requires the application of diverse -omics techniques on both short and long time-scales. Understanding the details of expression and epigenetic changes associated with changes of behavioral state will require interrogating natural and experimental populations with diverse -omics techniques. Such sampling may well turn out to be a major limiting factor for many studies, since researchers studying bird behavior are often more hesitant to destructively sample individuals, especially those from long-term studies. Additionally, not all techniques are well standardized in wild animals and those approaches requiring euthanasia may be adopted slowly. Approaches such as comparative genomics, blood transcriptomics, and blood plasma proteomics will offer useful opportunities in many scenarios, such as when working with difficult-to-access or threatened species. On the other hand, in less restrictive situations, invasive approaches have already yielded exciting new insights, such as recent work in brain transcriptional profiles studied in birds behaving in the wild .A phylogenetic framework is essential to understanding the evolution of CB , but hasUnderstanding the function and molecular mechanism of CB will help clarify a major topic in evolutionary biology that has intrigued scientists for decades: how can natural selection favor non-breeding individuals who help raise others’ offspring? Some have argued that CB is not an adaptive trait or evolutionarily stable strategy  but a fuThe sociogenomics of social behaviors such as CB and parental care in birds is still in its infancy. The functions, mechanisms, and the genomic architectures of these behaviors are fertile avenues to be understood with high-throughput techniques in combination with data collection in laboratory and field settings. Complex traits like CB act as a conceptual hub that brings together many fields, from neuroscience to genomics and behavioral ecology. Diverse technologies and experimental approaches will be required for a full comprehension of the evolution and mechanisms underlying CB in birds.All authors planned the organization and themes of the manuscript. F.T.-G. led the writing of the manuscript, with extensive contributions from all authors. All authors edited and approved the final manuscript.


The globe is presently reliant on natural resources, fossil fuels, and crude oil to support the world’s energy requirements. Human exploration for oil resources is always associated with irreversible effects. Primary sources of hydrocarbon pollution are instigated through oil exploration, extraction, and transportation in the Arctic region. To address the state of pollution, it is necessary to understand the mechanisms and processes of the bioremediation of hydrocarbons. The application of various microbial communities originated from the Arctic can provide a better interpretation on the mechanisms of specific microbes in the biodegradation process. The composition of oil and consequences of hydrocarbon pollutants to the various marine environments are also discussed in this paper. An overview of emerging trends on literature or research publications published in the last decade was compiled via bibliometric analysis in relation to the topic of interest, which is the microbial community present in the Arctic and Antarctic marine environments. This review also presents the hydrocarbon-degrading microbial community present in the Arctic, biodegradation metabolic pathways (enzymatic level), and capacity of microbial degradation from the perspective of metagenomics. The limitations are stated and recommendations are proposed for future research prospects on biodegradation of oil contaminants by microbial community at the low temperature regions of the Arctic. Arctic crude oil resources have become the major interest for exploitation to power the petroleum and other related industries ,2. A groThe degradation rate of hydrocarbon derivatives in the Arctic sea ice, marine, and soil are not feasible enough due to the cold temperatures combined with the perilous environment, reduced solubility, and high viscosity of hydrocarbons. An emergency mechanical technique was implemented to clean up almost 11 million gallons of oil from the infamous Exxon Valdez tanker oil spill, which displayed some drawbacks. The incident left many habitats and wildlife species still recovering and on the verge of being endangered ,8. PhysiThe bibliometric approach analyzes and evaluates research publications by monitoring the trajectory and development of the literature attributes in a scientific field. Interpreting and mapping a visualization network of research articles, helps to evaluate the productivity of authors, research groups, journals, institutions, countries, and determine the prospective state of knowledge of the field of study ,12. ThusAdditionally, a literature analysis was carried out to present the various aspects of microbial degradation of hydrocarbons, particularly in the Arctic marine region, since this was hardly reported compared to the Antarctic. The literature analysis compiled the consequences of hydrocarbon pollution in the Arctic marine environment, followed by the application of diverse microbial communities to degrade hydrocarbons, the metabolic pathway of the oil-degrading microbial community at enzymatic levels, microbial degradation capacity at the metagenomic level, and future prospects of bioremediation in the Arctic marine environment.Comprehensive data mining and analysis of scientific publications for a literature review are the foundation to establish and solve a problem in every aspect of research. A researcher should be able to grasp and understand the overview of a particular field of research. Bibliometrics is a statistical approach used to analyze scientific research or review articles, books, and other publications, which is often referred to as scientometrics ,14. ThisThe present emerging trends in hydrocarbon pollution by microbial communities in cold marine environments are important to evaluate the advancement and dynamics in bioremediation research. This section provides a bibliometrics analysis on the existing literature published during the last 10-year period (from 2009 to 2019). A comparative analysis was performed to highlight the application of microbial communities native to the Arctic and Antarctic cold marine environments in degrading hydrocarbon contaminants. A comparison between both polar regions provides better insights and overview of the proposed study.The analysis and evaluation of current trends were employed through a data searching of the “Scopus” database produced by Elsevier . This poThe process of document collection in relevance to the hydrocarbon degradation by Arctic and Antarctic marine microbial communities as the main theme is represented in the flowchart . The keyAnalysis of co-occurrence of keywords is a bibliometric technique that determines the association between topics in the research field of interest. The VOS viewer software constructed visualizing bibliometric maps based on the bibliographical, citation, and all keywords information of 175 and 144 articles that were exported separately. This tool is useful in finding the relatedness or connections between different items or pairs that exhibit specific link strength. The larger the value  of a link, the greater the strength of a link . Figure The findings showed that the term “bioremediation” was most frequently encountered and was established as the central theme of this topic. The keywords including “microbial community”, “biodegradation”, and “hydrocarbons” were profoundly associated with “bioremediation”. The use of microorganisms to remove or degrade environmental pollutants in oil spill incidents that threaten the marine ecosystem defines bioremediation ,24. BiolThe pattern in the number of publications published on the topic of oil spill remediation or hydrocarbon degradation in cold marine environments with the application of indigenous microbial communities has been extensively increasing from 2009 to 2019 . Many reThe published journal articles in relevance to hydrocarbon degradation by microbial community in cold marine Arctic and Antarctic environments were classified in various subject areas. This highlighted three main areas of knowledge b, which Bibliometric parameters such as countries and affiliated institutions provide opportunities for an international level of collaborations in the particular research field. This also helps some researchers to pursue their research career in established institutions or countries related to their specialization. The trends of publications were analyzed over the last decade based on the 144 and 175 articles related to hydrocarbon pollution in the Antarctic and Arctic marine environments and are presented in a global map . The mosIn addition, the most progressive research affiliated institutions in publishing journal articles are listed in The trends of preferred journals from 2009 to 2019 were analyzed based on their number of publications on the topic of hydrocarbon biodegradation through the application of microbial communities in the Arctic and Antarctic marine environments . Figure The literature search from the database of 144 documents showed an extensive publishing by the top 10 journals in the study of hydrocarbon degradation by microbial communities from the Antarctic marine environment. According to In addition to this, the Cite Score (Elsevier-Scopus), Clarivate Analytics of Journal Citation Reports (JCR), and Impact Factor (IF) information were evaluated based on the statistics of number and quality of documents and source journals. According to the Cite Score in 2019, nine journals  and eighLeading authors in publishing relevant articles were analyzed from the Scopus database based on the bibliometric parameters including author (Scopus) ID, current affiliations, highest cited document, total citations and publications, h-index, journal, and cited by. The total number of publications  on the study of hydrocarbon degradation by microbial community in the Arctic and Antarctic marine environments are summarized in Based on This study recognizes several shortcomings that could be explored in further research. The results of the bibliometric analysis are characterized by the research area, the selected timeframe, database, keywords and articles analysis, and the evaluated variables. Therefore, comparisons with multiple data outputs such as Web of Science and Scopus will be effective to find notable differences to assess the latest research trends ,31. In tOur bibliometric analysis showed that the research output in the bioremediation or biodegradation of hydrocarbons by Arctic marine microbial communities outnumber those for the Antarctic marine environment. Subsequently, an extensive literature analysis will identify various aspects of microbial degradation of hydrocarbons, from the fate of oil spills in the ocean to the enzymes or genes that are responsible for biodegradation . Oil conIn the Arctic, the primary sources of hydrocarbon pollution generally come from offshore drilling, oil tank blowouts or leaks, fuel transportation, leakage from ships, pipeline leakages, runoffs from land or river, discharges from water, and long-range transportation of pollutants via water. The marine ecosystem is largely affected, especially by large-scale oil spill disasters such as the Exxon Valdez tanker oil spill incident in Alaska (1989), Deepwater Horizon oil rig explosion in the Gulf of Mexico (2010), and Norilsk diesel fuel spill in Russia (2020). Such disasters were caused by human errors, illegal disposal of oil waste, and bunkering of oil among the ships . There w6 m2 of slick can be formed from 1000 kg of oil spilled, which depletes the oxygen level and causes pH changes in the seawater due to the disruption of gaseous (oxygen and carbon dioxide) exchange [The oil hydrocarbons spilled on marine surfaces encounter several weathering processes including photo-oxidation via carbon dioxide and water, emulsification, dissolution dispersion, evaporation, sedimentation, adsorption, as well as microbial biodegradation ,45. Oil exchange ,47.Accidental or unnecessary waste discharges associated with offshore drilling such as production water may be deposited in the sediments of the marine environment. Moreover, to sustain the pressure and rushing of the hydrocarbons into productions wells, tonnes of injection water are introduced into the system. Despite clean-up efforts that are based on regulatory guidelines, some of the low molecular weight hydrocarbons, inorganic salts, additives, monocyclic aromatic hydrocarbon (BREX), polycyclic aromatic hydrocarbons (PAHs), and other aromatic compounds remained in the environment . Apart fThe integrity of the Arctic’s ecosystem, including marine mammals, birds, corals, fish, and wildlife at coastal and shore regions have been destroyed in the pursuit of natural gas and crude oil exploration. The wildlife, marine animals, and birds manifested behavioral changes in their motility, feeding, and physical activities and were affected by disrupted growth and reproduction. Most of the hydrocarbons are less toxic when present at low amounts, although some aldehydes with low molecular weight, (monocyclic aromatic hydrocarbon) MAHs, and (polycyclic aromatic hydrocarbon) PAHs are stable, extremely carcinogenic, and toxic . The clePimephales promelas) [Anguilla anguilla) [Calanus finmarchicus and C. glacialis); damaged deoxyribonucleic acid (DNA) and inhibition in acetylcholine esterase (AChE) observed in Littoral crab (Carcinus aestuarii) [Chlamys islandica), blue mussel (Mytilus edulis), and red king crab  that led to nervous system damage, behavioral changes in swimming to escape, and induced oxidative stress [Various fish are affected by PAHs and their derivatives, including naphthalene that was observed to reduce the larval growth of fathead minnow (romelas) ; inducednguilla)  and Arctstuarii) , Icelande stress ,55,56.Delphinapterus leucas) [In marine mammals, PAHs induces oxidative enzyme activity in beluga whales ( leucas)  and an o leucas)  was yet  leucas) ,59. Mean leucas) .Oil spills or pipeline leakages absorbed by the soil at the shorelines resulted in reduced soil fertility that inhibits the growth of some plants. However, oil spreads minimally in soil, as hydrocarbons transform through blending in soil or snow ice, photo-oxidation, and microbial degradation . Plant lThis section discusses the applications of microbial community that focus mainly on the Arctic environments in degrading or remediating oil pollutants. The treatment of hydrocarbon contaminants, a combination of both mechanical and biological techniques or either one, are used to contain and remediate oil spills in the Arctic soil and ocean. The remoteness of the Arctic environment and its unpredictable conditions make oil spills extremely dangerous to animal habitats. Indigenous microbial floras concentrated in the seabed close to natural seeps have been found to degrade huge volumes of oil spills from the Deepwater Horizon and Exxon Valdez incidents at low temperatures and as deep as 1000 m . PhysicaPsychrobacter, Pseudoalteromonas, and Pseudomonas spp. [Marinobacter [Oleispira at 4 °C [The nature of hydrocarbon compounds is not viable for degradation, especially in low temperatures. Therefore, the factors including temperature, pH, nutrients, and oxygen can influence the microbial activity . Psychronas spp. ,72 are pnobacter , exhibit at 4 °C .Sphingomonas strains that can adapt to various nitrogen concentrations [Biostimulation, which is the incorporating of nutrients for hydrocarbon degradation, stimulates the nitrogen content for assimilation and induces growth. The oil-contaminated soils in the Arctic have low levels of nitrogen concentrations due to an increase in carbon, which limits the bioremediation process. The carbon sources essential for degradation are supplemented through hydrocarbon and water supplying oxygen and hydrogen ,76. Thustrations ,78.Phialophora spp. and Hormoconis resinae [The addition of hydrocarbon-degrading bacteria, algae, fungi, algae, or yeast to prevailing population of microbes is known as bioaugmentation, which is inexpensive and allows in-situ bioremediation. The oil-degrading microorganisms isolated from oil spill sites increase the degradation capacity of organic pollutants in conjunction with indigenous microbiota, with high tolerance towards the toxicity of hydrocarbons ,80. The  resinae ,88 are k resinae ,90,91.Complex hydrocarbon compounds in nature degrade according to the following order of biodegradability (least to most): linear alkanes—branched alkanes—low molecular weight alkyl aromatics—monocyclic aromatic hydrocarbons (MAHs)—cyclic alkanes—polycyclic aromatic hydrocarbons (PAHs)—high molecular weight asphaltenes ,95. AromPseudomonas, Dietzia, Pseudoalteromonas, Halomonas, Cycloclasticus, and Marinomonas found in the Arctic Ocean, which were responsible for PAH degradation. Cold-acclimated microorganisms such as PAHs-degrading Pseudomonads (β-Proteobacteria) and Sphingobium sp. C100 (α-Proteobacteria) were enriched in the deep-sea sediments of the Arctic Ocean [Marinomonas profundimaris D104 [Colwellia was present in the Svalbard sea ice of the Arctic [Gammaproteobacteria and Bacteroidetes are the predominant groups underneath the shore-fast ice of the Arctic sub-ice, while Alphaproteobacteria majorly dominates the surface of seawaters [The petrogenic origins of PAHs were identified in the Arctic sea through an extensive sampling with depths of 367–4320 m, while some low molecular weight PAHs were abundant in the Arctic Ocean North–South Transect, notably close to the land . Moreoveic Ocean , Marinome Arctic ,102. Theeawaters .Hydrocarbon degradation involves two common pathways: aerobic (oxygen-dependent) and anaerobic (oxygen-independent) . Microbi2) molecule as an electron acceptor through the introduction of one or two atoms of O2 to a substrate (hydrocarbon) [Analysis of potential microbial community of PAHs degraders found that they utilized genes encoding for the PAH monooxygenase or dioxygenase , which could be utilized as biomarkers . These eocarbon) .Apart from that, anaerobic biodegradation of hydrocarbons requires compounds other than oxygen that can act as electron acceptors such as sulphate, nitrate, and ferric chloride . A propoMicrobial communities possessing high enzymatic capacity can degrade complex hydrocarbons through transformation of contaminants into less toxic forms. The distinctive catalytic properties evolved in the psychrophilic microbial community express cold-adapted enzymes that have enhanced flexibility, reduced thermal stability, and greater catalytic effectiveness in extremely cold environments ,119. TheThe rate and magnitude of hydrocarbon biodegradation in cold marine environments are considered to be crucial elements and require understanding in the removal of compounds by microbes. Multiple aspects such as pollutants, oxygen, pH, nutrient, temperature, and the wide range of microbial community have been found in the contaminated regions. Microbial communities present in the low temperatures of Arctic marine ecosystems are capable of degrading hydrocarbons as a carbon source and promoting energy production while utilizing the bioavailability of nutrients such as nitrogen . The molRecent advancements in analyzing and understanding the metagenomics of the microbial communities are essential to access the taxonomic and composition of functional genes in the biodegradation of oil pollutants. The study of genetic material of intact communities is known as the metagenomics process. Metagenomics involve the use of next-generation sequencing (NGS) with the latest approaches including whole-metagenome sequencing (WMS) ,126. TheThe application of metagenomics in the largest oil spill accident to-date, the 2010 Deepwater Horizon drilling rig explosion, provided an opportunity for researchers to explore and deepen the knowledge of the biodegradation of hydrocarbon pollutants. PAH-degrading bacteria such as Cycloclasticus , MarinobEmerging technologies in bioremediation strategies have been introduced with the main purpose to restore contaminated ecosystems in an environmentally sustainable and cost-effective manner. The ecosystem in the extreme Arctic conditions requires a longer time to recover from pollution . Ex-situBioaugmentation is one of the in-situ bioremediation techniques that is less common in the Arctic due to limited nutrients levels that inhibit successful bioremediation of hydrocarbon contaminants. Therefore, the addition of nitrogen, carbon, and phosphorous sources is essential to optimize the growth of microbial communities and increase the efficiency of biodegradation of hydrocarbons ,138. BioThe recent developments in biotechnology and engineering have produced new technologies to deliver novel and effective methods in managing polluted Arctic marine environments. Bibliometric analysis is valuable to identify the gaps of knowledge in the field of study. Polar regions are immensely vulnerable to rising anthropogenic activities that pose risks to the environment because of the high possibility for the next major oil pollution to occur. Bioremediation approaches improve the sustainability of the environment via in-situ remediation, including applications of native cold-acclimated microbial communities that safely degrade harmful hydrocarbons. The investigations on biodegradation of hydrocarbons in cold regions remain limited, and more information through enzymatic and metagenomics insights are required to respond and prepare towards the consequences of oil pollution in the Arctic marine ecosystem. Over the years, studies had proven that biodegradation of hydrocarbon via indigenous microbial communities is able to enhance the remediation process. This review has provided an overview for further scientific research and highlighted future research that are needed for microbial applications in the bioremediation of oil pollutants.


Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics. In this review, we summarize the advances in gene-editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated with the Cas9 and Cpf1 proteins. These tools support great opportunities for the future development of plant science and rapid remodeling of crops. Furthermore, we discuss the brief history of each tool and provide their comparison and different applications. Among the various genome-editing tools, CRISPR has become the most popular; hence, it is discussed in the greatest detail. CRISPR has helped clarify the genomic structure and its role in plants: For example, the transcriptional control of Cas9 and Cpf1, genetic locus monitoring, the mechanism and control of promoter activity, and the alteration and detection of epigenetic behavior between single-nucleotide polymorphisms (SNPs) investigated based on genetic traits and related genome-wide studies. The present review describes how CRISPR/Cas9 systems can play a valuable role in the characterization of the genomic rearrangement and plant gene functions, as well as the improvement of the important traits of field crops with the greatest precision. In addition, the speed editing strategy of gene-family members was introduced to accelerate the applications of gene-editing systems to crop improvement. For this, the CRISPR technology has a valuable advantage that particularly holds the scientist’s mind, as it allows genome editing in multiple biological systems. Oryza sativa (rice), Triticum aestivum (wheat), Zea mays (maize), and Glycine max (soybean), will increase by just 38–67% ..ARGOS8 v(SIAGL6) gene in tomato. Knockout of the SIAGL6 gene improved the fruit setting of tomato under heat stress . F. F197]. 0 generation by engineering a genome-wide mutant library in rice. Upon consideration of the highly efficient editing achieved in the T0 generation, CRISPR/Cas9 was used to engineer a genome-wide mutant library in rapeseed, which will promote gene characterization and its beneficial applications at a later stage.The adopted CRISPR system and its usage will promote the rapid progress of crop breeding and functional genomics. Recently, new and versatile breeding technologies have been implemented to facilitate the engineering of multiple genetic loci in different breeding varieties, which will improve food security and strengthen crop amelioration. Moreover, the perusal of the literature for genomic sequences and their functions is a prerequisite for efficient genome editing. In the future, we will likely witness the increased use of CRISPR for clarifying genomic structures and their role in plants, such as the transcriptional regulation of Cas9 and Cpf1, the monitoring of genetic loci and mechanisms, and the regulation of promoter activity. Moreover, it will also include the modification and identification of epigenetic behavior in communicating the stable relationships between single-nucleotide polymorphisms (SNPs), which are investigated by genetic traits, and genome-wide association studies. Interestingly, this technique was designed to achieve phenotypic characterization in the TThe CRISPR technology can classify any new crop traits in the category of plant synthetic biology . In our Genome editing is becoming the most used and versatile tool for crop improvement and functional genomics. The attractive survival landscapes, such as the efficiency, multiplexing, integrity, and simplicity, as well as the highly specific nature, of the genome-editing technologies mentioned here indicate the manner in which crop breeding is carried out and pave the way for plant breeding for the next generations. This new strategy for crop improvement has proven to be efficacious based on a review of the literature on transcriptomics, biotechnology, genomics, and phonemics. The regulation of transgenic crops was also coherently simplified to support the rapid progression of this technology and render these crops acceptable for consumer usage. In addition to these social and technical challenges, the CRISPR technology was used for the first time to edit plant genomes. Therefore, the use of genome editing on a large scale for crop improvement is already a reality. The journey of genome editing raises ethical questions that need to be addressed by researchers and society on a massive scale.


Listeria monocytogenes is a Gram-positive bacterium, mostly known as a food-borne pathogen, capable of causing life-threatening and often fatal infections. However, since in the majority of cases the human immune system is able to mount potent innate and adaptive immune responses that control infections by Listeria monocytogenes, the microorganism has become an attractive vector for the development of cancer vaccines. The review by Flickinger Jr., Rodeck and Snook  on the use of Listeria monocytogenes as a vector for cancer immunotherapy is described and commented here.Cancer is a wide group of diseases, which was responsible for 9.6 million deaths in 2018. Cancer immunotherapies have become a reality, with the first approval for sipuleucel-T for prostate cancer therapy occurring in 2010.  Cancer is a wide group of diseases that can start in virtually any organ or tissue by abnormally growing cells that invade other parts of the body, ultimately leading to death. According to World Health Organization (WHO) data, cancer was the cause of 9.6 million deaths in 2018 . In the last years, the cancer immunotherapy field has rapidly evolved. Immunotherapies can be classified as passive and active therapies. Passive therapies encompass the administration of monoclonal antibodies, oncolytic viruses and donor-activated lymphocytes activated towards cancer cells . In contClostridium, Bifidobacterium, Salmonella, Mycobacterium, Bacillus and Listeria have been reported to target cancer cells and to act as anticancer agents [The development of bacterial vaccines against cancer is a highly active research field, with expected applications in the near future. The use of bacteria as anticancer agents was described for the first time in 1890 by the New York Memorial Hospital surgeon William B. Coley . Since tr agents . Listeria monocytogenes is a Gram-positive bacterium, mostly known as a food-borne pathogen, capable of causing life-threatening and often fatal infections, including bacteremia, encephalitis and sepsis [Listeria monocytogenes, turning the microorganism into an attractive vector for the development of cancer vaccines. In their paper entitled “Listeria monocytogenes as a vector for cancer immunotherapy: current understanding and progress”, Flickinger Jr., Rodeck and Snook present a thorough and elegant review on the use of Listeria monocytogenes as a vector for immunotherapies against cancer [Listeria monocytogenes as a vector for cancer immunotherapies, namely the possibility of a repeated administration of the microorganism to boost T-cell responses, the ability of the bacterium to induce both innate and adaptive immune responses, and its ability to act as a potent stimulator of cell-mediated immunity and cytotoxic lymphocytes. After briefly reviewing the main molecular mechanisms used by Listeria monocytogenes to cause human infections, Flickinger Jr., Rodeck and Snook review and discuss the development of attenuated strains suitable for vaccination, focusing on strategies involving the deletion of virulence genes, the construction of strains that ectopically express virulence or metabolic genes, and of strains that are killed but metabolically active. Various strategies used for the genetic engineering of strains expressing the virulence factors ActA and LLO, by fusing them with tumor-associated antigens, are also presented [Listeria monocytogenes as a vaccination vector. The perspectives presented by Flickinger Jr., Rodeck and Snook for future developments of Listeria monocytogenes-based vaccines point out the targeting of angiogenesis proteins and the use of strategies combining classical approaches like radiation and chemotherapy with Listeria monocytogenes vaccines [Listeria monocytogenes cancer vaccines are immense, and there is great hope that such vaccines will contribute to innovative anti-cancer therapies that benefit cancer patients.d sepsis . The orgd sepsis . Nevertht cancer . In the resented . The autvaccines . The pos


This article will pull together a comprehensive assessment of the association of cardiovascular disease, diabetes, obesity and COVID-19, its impact on the health systems and how best health systems can respond to mitigate current challenges and future needs. We anticipate that in the context of this pandemic, the cardiovascular disease and diabetes patients need a targeted strategy to ensure the harm to this group does not translate to huge costs to society and to the economy. Finally, we propose a triage and management protocol for patients with cardiovascular disease and diabetes in the COVID-19 settings to minimize harm to patients, health systems and healthcare workers alike.Patients with cardiovascular disease and diabetes are at potentially higher risk of infection and fatality due to COVID-19. Given the social and economic costs associated with disability due to these conditions, it is imperative that specific considerations for clinical management of these patients be observed. Moreover, the reorganization of health services around the pandemic response further exacerbates the growing crisis around limited access, treatment compliance, acute medical needs, and mental health of patients in this specific subgroup. Existing recommendations and guidelines emanating from respective bodies have addressed some of the pressure points; however, there are variations and limitations  On March 11th, 2020, coronavirus disease 2019 (COVID-19) was designated as a global pandemic by the World Health Organization (WHO). As of 28th May, 5,716,271 confirmed cases have been reported with ~356,124 deaths globally from 188 countries . ConversIndependent of other medical problems such as CVD, patients with diabetes are at elevated risk for infection from COVID-19 by 2-to-3 fold . This man = 407) at high risk of venous thromboembolism; and the high-risk patients who didn't receive prophylactic therapy (11%) developed venous thromboembolism  and diabetes , followed by CVD  . A recenembolism .2 . The requirement for ventilatory support was equal between the obese and non-obese patients . Obesity was associated with higher mortality rates in critical care when compared to normal or underweight COVID-19 positive patients . These data derive from the ICNARC case mix programme database. The case mix programme is the national clinical audit of patient outcomes from adult critical care coordinated by the ICNARC. For more information on the representativeness and quality of these data, we encourage readers to contact ICNARC  in the United Kingdom (UK) received notification of 2,621 COVID-19 positive cases requiring critical care . Analysit ICNARC .In the setting of COVID-19, specialist cardiologists and endocrinologists are confronted with a number of critical issues on management and treatment of CVD. There has been speculation regarding the risk associated with the use of ACE inhibitors (ACEi) and angiotensin receptor blockers (ARBs) in patients with COVID-19 , 24. ThiPoor glycaemic index is known to cause immune suppression through impaired neutrophil degranulation, deficient complement system and phagocytosis . The co-Healthcare workers responsible for the care of patients with diabetes and cardiovascular disorders infected with COVID-19 face threats to their own well-being, being at risk of exposure to a high viral load . Time isRefugees, undocumented immigrants and members of aboriginal communities also have limited provisions of access and medical relief in pandemic situations, due to structural factors and poor socioeconomic conditions that put them at compounded risk due to cardiovascular and diabetes comorbidities.Professional societies such as the American Association of Clinical Endocrinologists  and EuroNHS clinical guidelines for the management of diabetic patients in COVID-19 recommend expedition of treatment and discharge of inpatients, and the use of virtual clinics and teleconsultations in primary and secondary care settings . HoweverCurrent approaches to the management of this population aim to continue care of patients during COVID-19, while minimizing the risk of transmission to both healthcare workers and patients. The current protocol at academic medical centers in China for Acute Myocardial Infarction involves compulsory screening for fever and respiratory symptoms, and any patients with STEMI that have suspected or confirmed infection are treated with emergency intravenous thrombolysis, in the absence of contraindications . For carWith regard to the use of ACEi and ARBs, several societies have highlighted that due to the limited nature of the evidence on this matter, it is advisable that ongoing management with such medication may continue in patients with diabetes and hypertension, unless otherwise clinically contra-indicated as per the case profile –48. Ongotorsades de pointes) and sudden cardiac death . This may lower the risk of infection to healthcare workers and patients. Cardiovascular experts may brace themselves for deployments in different settings, for limited, extended or repurposed causes. The mobility of staff between COVID-19 treatment units and other patient facing consultation should be limited to avoid opportunities for nosocomial transmission. COVID-19 and patients with CVD, diabetes or obesity impact each other in compounding and negative dimensions. These patients are at increased risk of COVID-19 related hospitalization, morbidity and death; and those with COVID-19 also show propensity to increasing and emergent acute cardiovascular events. It is important to identify COVID-19 patients who are at high risk of VTE so that appropriate prophylaxis treatment could be initiated , 59. Ant2 should be used as a prognostic indicator of mortality in critical care settings due to COVID-19 (2) with severe COVID-19  might mislead or compromise the safety of obese people at lower BMIs , washing hands, social distancing. A New England Journal of Medicine study showed efficacy of face masks in preventing further transmission of Coronavirus from symptomatic individuals . It is eRecently the CDC recommended wearing cloth face coverings in public settings where other social distancing measures are difficult to maintain , especially in areas of significant community-based transmission . CoronavPandemics like COVID-19, SARS and Spanish flu invoke irrational and heightened fear which could be linked to incidents of xenophobia and discrimination . A publiPatients with comorbid CVD, diabetes and obesity are potentially vulnerable in a pandemic –70. It mThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.†The COVID19 pandemic is causing an unprecedented public health crisis impacting healthcare systems, healthcare workers and communities. The COVID-19 Pandemic Health System REsilience PROGRAM (REPROGRAM) consortium is a think-tank of leading international healthcare physicians, researchers and policymakers formed to champion the safety of healthcare workers, policy development and advocacy for global pandemic preparedness and action.SBh devised the project, the main conceptual ideas and proof outline. SBh and AR wrote the first draft of the manuscript. SBh encouraged AR to investigate and supervised the findings of this work. All authors discussed the results and recommendations, and contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The impact of emotion regulation interventions on wellbeing has been extensively documented in literature, although only in recent years virtual reality (VR) technologies have been incorporated in the design of such interventions, in both clinical and non-clinical settings. A systematic search, following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, was therefore carried out to explore the state of the art in emotion regulation interventions for wellbeing using virtual reality. The literature on this topic was queried, 414 papers were screened, and 11 studies were included, covering adults and older adults. Our findings offer an overview of the current use of VR technologies for the enhancement of emotion regulation (ER) and wellbeing. The results are promising and suggest that VR-based emotion regulation training can facilitate the promotion of wellbeing. An overview of VR-based training interventions is crucial for better understanding how to use these tools in the clinical settings. This review offers a critical debate on the structure of such intervention protocols. It also analyzes and highlights the crucial role played by the selection of the objective and subjective wellbeing assessment measures of said intervention protocols. Emotion is a cultural and psychobiological adaptation mechanism that allows each individual to react flexibly and dynamically to environmental contingencies . EmotionThe growing development of new technologies and the interest in applying them in the field of psychology have led to the development of novel virtual reality (VR) systems for neuro-rehabilitation , or the Health and wellbeing are considered as indispensable resources for societies and human development . The WorA systematic review of the scientific literature has been performed to identify studies that reported VR-based ER interventions for wellbeing in healthy and clinical adults and examined the structure of their protocols. The methodology is presented in the following paragraphs. Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines were followed . Four hiThis systematic review aims to evaluate the wellbeing and emotion regulation outcome of VR-based interventions in adults and older adults without psychopathological conditions. Given that the interest in VR continues to grow, researchers must focus on how the characteristics of VR systems and the different aspects of the training tasks could influence the intervention outcomes. The aim of this review is to provide knowledge and guide researchers in the selection of the most appropriate VR experience for ER interventions. The flow chart of the search strategy results, according to the PRISMA flow diagram, is shown in The present systematic review considered randomized control trials, nonrandomized control trials, intervention studies, and case-control studies. Studies on emotion regulation for wellbeing with virtual reality (VR) devices in healthy or pathological adults and older adults presenting the following clinical conditions: traumatic brain injury, motor disabilities, tumor, chronic conditions (heart failure and chronic pain), were included. The review only includes studies in the English language, and which satisfied strict criteria for eligibility . Articles that treat psychopathological disorders such as post-traumatic stress disorder, phobias, substance abuse or psychosis, or lacked necessary information for review in the full-text or the abstract were excluded. Reviews, meeting abstracts, proceedings, poster presentations, notes, case reports, letters to the editor, assessment protocols, editorials, and other editorial materials were also excluded. Retrospective studies were not included because the area of interest requires post-intervention outcomes.To assess the risk of bias, the reviewers followed the methods recommended by The Cochrane Collaboration Risk of Bias Tool  and the Of 414 non-duplicate studies, 386 did not fit the preliminary inclusion criteria; specifically, they did not present ER interventions for wellbeing using VR systems in adult and older adult populations. Subsequently, the full text of 28 articles was retrieved and the studies were evaluated for the specific inclusion criteria. Of 28 studies, only 11 passed the full-text screening phase, while 17 studies were excluded for the reasons that follow: Not interventions (= 4); Results not reported (= 7); Qualitative/descriptive study (= 6). The present flow chart  shows a The majority of the studies except one  exhibiteSeveral investigations suggest that VR-based ER interventions in adults and older adults could improve quality of life, physical and mental health and delay the onset of health disorders ,30. We cIn the following paragraphs we discuss the characteristics of the selected VR interventions. The examined studies focus on the use of virtual environments for intervening on emotion regulation processes, and for improving the wellbeing of healthy and clinical populations.The evaluated interventions for adults and older adults  are summarized in In what follows we examine studies conducted in a healthy population see . VillaniWeerdmeester et al.  have exaBornioli et al. ,50 suppoOther studies have used interventions on patients with specific pathologies see  using vi® Wii Fit [The examined studies have used different types of virtual reality systems. Among the studies that have used immersive VR systems, two have selected a head-mounted display in combination with a smartphone and a head-tracker ,44. Howe Wii Fit ; tablet  Wii Fit ,46,49,50 Wii Fit . Positive emotions have a significant influence on mental and physical health ,57. TheiA large number of studies among those evaluated in this systematic review use physiological markers as an objective measure to assess the effectiveness of VR interventions for ER. In this regard, the appraisal theory of Scherer allows us to explain the link between emotional states experienced by participants during interventions and their physiological responses ,62. In pIn addition to the studies examined above, a neurofeedback (NFB) system had been used to enable the participants to regulate their brain activity, to enhance and recover emotional and cognitive capacity and to improve their underlying neurobiology . NeurofeGiven the primary objective of the present manuscript, in The Social Care-Related Quality of Life (SCRQoL) Scale was administered to track changes in the quality of life of older users to their daily routines, based on the outcome domains of social care-related quality of life identified in the Adult Social Care Outcomes Toolkit (ASCOT) . MoreoveThis review aimed to investigate how emotion regulation interventions using virtual reality (VR) systems can enhance the wellbeing in healthy and clinical adults and older adults, without presenting psychopathological conditions. In recent years there is a growing interest in the use of advanced technologies in supporting wellbeing and promoting health , followiIn the examined studies, virtual reality has been used in different ways to promote wellbeing in healthy and clinical populations. Some authors used mindfulness as a non-pharmacological approach to manage participant’s emotion regulation abilities and to enhance their wellbeing ,44. It hIn the present review, the combination of VR with biofeedback systems was particularly interesting. In recent years the development and use of game-based biofeedback to promote physical and mental wellbeing is growing ,77,78. ANeurofeedback (NFB) is a type of biofeedback which facilitates the real-time voluntary regulation of brain activity through a brain-computer interface ,68. LoreFor instance, Singh et al.  suggest These results are an important outcome for wellbeing. The literature demonstrates that VR-based interventions are as effective as traditional ones in the treatment of different mental disorders, but can achieve positive outcomes in less time . The difWhy are VR-based ER interventions important for wellbeing? To address this question, we have to consider several things. Emotions can be transient phenomena which emerge from momentary situational goals and subside with the short-term achievement of such goals . EmotionThe present review underlines that virtual reality systems evoke a general positive emotion and could promote a healthy life  and an oThe obtained results show that interventions using virtual reality systems allow people to change or improve their ER strategies. One such strategy is “situation selection” . It is aOne of the main aims of emotion regulation interventions is to modify the emotional responses of the subjects to one specific situation . The outIn conclusion, the choice of wellbeing measures is essential to assessing the efficacy of the intervention. Humans do not act mechanically, but rather according to their subjective interpretation of the world. Hence, objective indicators alone may not be sufficient to hedge the several conceptualizations of wellbeing . It is fIn conclusion, the results of this systematic review show that technology can improve the ability of people to handle emotionally-rich life situations by training more contextually adaptive emotion regulation strategies.The use of virtual reality in this sense is promising because it allows the user to learn complex ER strategies in the context of life-like digital environments. VR interventions can modify the user’s ER by inspiring new actions, allowing for the modification of the emotional response across a reappraisal of emotional stimuli, and subsequently, memorizing the re-evaluated experience. Finally, virtual reality is a tool that fosters a sense of environmental mastery, and, a feeling of personal growth and autonomy. The literature in this field is going in an interesting direction, and we recommend some future steps based on the findings of this systematic review, for future design, implementation, and evaluation of VR-based ER interventions in healthy adults and older adults. The results of this systematic review underly the importance of an appropriate assessment and highlight the positive effects of assessing both subjective and objective measures in future studies to fully evaluate the efficacy of VR interventions for ER. The results from both subjective and objective measures will provide an overall and complete frame of the efficacy of VR interventions for ER. Finally, future research should aim to investigate the underlying mechanisms and factors that may contribute to the effectiveness of biofeedback systems when using VR interventions for ER in order to maximize their positive therapeutic outcomes.


Chest computed tomography (CT) is crucial for the detection of lung cancer, and many automated CT evaluation methods have been proposed. Due to the divergent software dependencies of the reported approaches, the developed methods are rarely compared or reproduced.The goal of the research was to generate reproducible machine learning modules for lung cancer detection and compare the approaches and performances of the award-winning algorithms developed in the Kaggle Data Science Bowl.We obtained the source codes of all award-winning solutions of the Kaggle Data Science Bowl Challenge, where participants developed automated CT evaluation methods to detect lung cancer . The performance of the algorithms was evaluated by the log-loss function, and the Spearman correlation coefficient of the performance in the public and final test sets was computed.Most solutions implemented distinct image preprocessing, segmentation, and classification modules. Variants of U-Net, VGGNet, and residual net were commonly used in nodule segmentation, and transfer learning was used in most of the classification algorithms. Substantial performance variations in the public and final test sets were observed (Spearman correlation coefficient = .39 among the top 10 teams). To ensure the reproducibility of results, we generated a Docker container for each of the top solutions.We compared the award-winning algorithms for lung cancer detection and generated reproducible Docker images for the top solutions. Although convolutional neural networks achieved decent accuracy, there is plenty of room for improvement regarding model generalizability. Lung cancer is one of the most prevalent cancers worldwide, causing 1.76 million deaths per year . Chest cDue to the improved performance of machine learning algorithms for radiology diagnosis, some developers have sought commercialization of their models. However, given the divergent software platforms, packages, and patches employed by different teams, their results were not easily reproducible. The difficulty in reusing the state-of-the-art models and reproducing the diagnostic performance markedly hindered further validation and applications.To address this gap, we reimplemented, examined, and systematically compared the algorithms and software codes developed by the best-performing teams of the Kaggle Data Science Bowl. Specifically, we investigated all modules developed by the 10 award-winning teams, including their image preprocessing, segmentation, and classification algorithms. To ensure the reproducibility of results and the reusability of the developed modules, we generated a Docker image for each solution using the Docker Community Edition, a popular open-source software development platform that allows users to create self-contained systems with the desired version of software packages, patches, and environmental settings. According to Docker, there are over 6 million Dockerized applications, with 130 billion total downloads . The DocWe obtained the low-dose chest CT datasets in Digital Imaging and Communications in Medicine format from the Kaggle Data Science Bowl website . The datTo systematically compare the solutions developed by the award-winning teams, we acquired the source codes of the winning solutions and their documentation from the Kaggle news release after the conclusion of the competition. Per the rules of this Kaggle challenge, the source codes of these award-winning solutions were required to be released under open-source licenses approved by the Open Source Initiative  in orderWe compared the workflows of the top 10 solutions by examining and rerunning their source codes. For each solution, we inspected all steps taken from inputting the CT images to outputting the prediction. We documented the versions of the software package and platform dependencies of each solution. where n is the number of patients in the test set, iy is 1 if patient i has lung cancer, 0 otherwise, and iŷ is the predicted probability that patient i has lung cancer [The Kaggle Data Science Bowl used the log-loss function to evaluate the performance of the models . The logg cancer . If the To investigate whether models with high performance in the public test set generalize to the images in the final test set, we computed the Spearman correlation coefficient of the log-loss in the two test sets. All analyses were conducted using R version 3.6 .We reproduced the results by recompiling the source codes and dependencies of each of the top ten solutions. Since the solutions used various platforms and different versions of custom software packages, many of which were not compatible with the most updated packages or mainstream release, we generated Docker images  to managIn addition to the training dataset provided by the Kaggle challenge, most teams used CT images and nodule annotations from other publicly available resources. Frequently used image preprocessing steps include lung segmentation and voxel scaling. Voxel scaling ensures that the voxels of images from various CT scan protocols correspond to similar sizes of physical space. Variants of U-Net , VGGNet After lung nodule segmentation, classification algorithms were employed to generate final cancer versus noncancer predictions. Most of the solutions leveraged existing ImageNet-based architecture and transfer learning ,27. All Most of the winning teams developed their modules with Keras and Tensorflow. Only one team used Pytorch (the top-performing team), Caffe, or Lasagne. All of the top 10 teams employed a number of python packages for scientific computing and image processing, including NumPy, SciPy, and Scikit-image (skimage). A summary of package dependencies is shown in To facilitate reusing the code developed by the top teams, we generated a Docker image for each of the available solutions. Our developed Docker images are redistributed under the open-source licenses chosen by the original developers . DetaileThis is the first study that systematically compared the algorithms and implementations of award-winning pulmonary nodule classifiers. Results showed that the majority of the best-performing solutions used additional datasets to train the pulmonary nodule segmentation models. The top solutions used different data preprocessing, segmentation, and classification algorithms. Nonetheless, they only differ slightly in their final test set scores.The most commonly used data preprocessing steps were lung segmentation and voxel scaling . For nodTo enhance the reproducibility of the developed modules, we generated a Docker image for each of the award-winning solutions. The Docker images contain all software dependencies and patches required by the source codes and are portable to various computing environments , which wSince it was difficult to compile and release a large deidentified chest CT dataset to the public, the public test set only contains images from 198 patients. Leveraging the 5-digit precision of the log-loss value shown on the leaderboard, one participant implemented and shared a method for identifying all ground truth labels in the public test set during the competition . SeveralThere are several approaches future contest organizers can take to ensure the generalizability of the developed models. First, a multistage competition can filter out the overfitted models using the first private test set and only allow reasonable models to advance to the final evaluation. In addition, organizers can discourage leaderboard probing by only showing the performance of a random subset of the public test data or limiting the number of submissions allowed per day. Finally, curating a larger test set can better evaluate the true model performance and reduce random variability . If dataIn summary, we compared, reproduced, and Dockerized state-of-the-art pulmonary nodule segmentation and classification modules. Results showed that many transfer learning approaches achieved reasonable accuracy in diagnosing chest CT images. Future works on additional data collections and validation will further enhance the generalizability of the current methods.


Enhanced pharmacy services have been identified as a mechanism to address medicines and drug-related problems. The aim of the study was to explore the perspectives of practicing pharmacists on the scope of pharmacy service provision in Pakistan. This qualitative study was conducted at the Department of Pharmacy, the Islamia University of Bahawalpur (IUB). Face-to-face, in-depth interviews were conducted with practicing pharmacists at the university who were undertaking postgraduate studies. All interviews were audio-recorded, transcribed verbatim, and analyzed using thematic analysis. A total of 13 pharmacists were interviewed. The analysis of data yielded four themes and 12 subthemes. The themes included the current scenario of pharmacy services, the benefits of pharmacy services, barriers to implementation of pharmacy services, and strategies to improve their delivery. Pharmacist participants reported that patient-oriented pharmacy services have not been properly implemented in Pakistan. Pharmacists appear to be undertaking only conventional roles at various levels within the healthcare system. The participants indicated multiple benefits of patient-oriented pharmacy services, including safe and effective use of medicines, minimization of drug-related problems, and financial benefits to the healthcare system. Based on the findings, policy-makers are required to take the necessary steps to overcome pharmacist-related and policy-related barriers associated with the implementation of patient-oriented pharmacy services in Pakistan. Over recent decades, there has been considerable worldwide increase in the morbidity and mortality associated with chronic diseases. This has been linked with lack of individualized patient care and consequent medication-related issues . To thisPakistan is a low and middle income country (LMIC) that has been recently ranked lower than all other South Asian countries at 152th out of 189 countries, with a Human Development Index (HDI) value of 0.560 . Over thIn Pakistan, the three year Bachelor of Pharmacy degree was first initiated in 1948 . HoweverDespite these ongoing government-level efforts, as well as literature and policy calling for the implementation of pharmacy services, lack of recognition of the full scope of pharmacy services has remained a significant impediment for pharmacists to gain a foothold as competent and recognized healthcare professionals in Pakistan . With veGiven the evidence that the recognition and implementation of advanced pharmacy services have helped address the void in public health across many developed nations , it is dThe study was conducted at the Department of Pharmacy, the Islamia University of Bahawalpur (IUB). The IUB is located in the district of Bahawalpur, the Punjab province of Pakistan. The University was established in 1925 and encompasses 45 departments and 74 disciplines. Courses are available in Engineering, Computer Science, Arts and Humanities, Life Sciences, Sports Science, and Pharmacy . In 1990Postgraduate students with part-time employment in the private sector drug retail outlets (community pharmacies) or public sector hospital pharmacies and who were on study leave for their postgraduate education were invited to participate in the study. To assure the reliability and competence of the informants, and ultimately the quality of the data gathered, participants with a minimum work experience of five years duration were eligible to participate in the study.In-depth, semi-structured, face-to-face interviews were used as part of a qualitative study design to explore pharmacists’ views on the scope of pharmacy services in Pakistan. The qualitative method strives to understand phenomena in depth, where little information has previously been generated and for which the aim is not to generalize .The interview schema was developed through an extensive literature search ,31,32 anThe data were collected from 1st May 2019 to 31st July 2019. Saturation point criteria were used to determine the point at which the sample size was deemed adequate. The study participants were selected through a two-stage process, involving purposive and convenience sampling techniques . In the The data were analyzed using the thematic analytic approach of Braun and Clarke . Six phaEthical approval was obtained from the Pharmacy Research Ethics Committee (PREC) at the Islamia University Bahawalpur . Written informed consent was obtained from the study participants after explaining the aims and objectives of the study. The identities of the study participants remain confidential by assigning identification codes to them. A pressure-free and trustworthy environment was maintained throughout the interview. The participants were given the freedom to raise concerns, skip any question, or even withdraw from the interview at any time during the study without giving reasons, although none chose to quit the interview and all the raised concerns were candidly answered.In this study, the phrase “renting out or leasing out license of pharmacist” refers to “a contract between two parties, the lessor (owner of asset) and the leasee (user), in which a non-qualified medical store/pharmacy owner (leasee) pays a signed amount to the pharmacist (lessor having a pharmacy degree and license) for the use of rights granted under that license.”A total of 13 pharmacists were interviewed. The average interview duration was 26.4 min . The mean age of the study participants was 36 years . The characteristics of the respondents are outlined in The analysis of data yielded four themes and 12 subthemes. The themes included current scenario of pharmacy services, benefits of pharmacy services, strategies to improve pharmacy services, and barriers to the implementation of pharmacy services.All respondents were familiar with the term “pharmacy services”. According to respondents, these are the services offered by pharmacists directed at patient care. The majority of participants had an understanding of the terms dispensing, counselling, distribution, prescription monitoring, storage, procurement, and pharmacovigilance (12 out of 13 respondents). However, most of the pharmacists were unaware that domains such as pharmaco-economics (8 out of 13 respondents), posology (11 out of 13 respondents), and pharmaco-epidemiology (7 out of 13 respondents) were also part of sanctioned pharmacy services according to the DRAP Act 2012.Most of the pharmacists (10 out of 13 respondents) were of the opinion that foundation services, such as dispensing, storage, procurement, and sale of medicines, are currently being offered by pharmacists in Pakistan. Almost 70% of pharmacist respondents (9 out of 13 respondents) reported that counselling is of low quality and was being performed properly at a few private hospitals but not government hospitals. The same proportion of pharmacists (9 out of 13 respondents) were of the opinion that prescription monitoring, pharmacovigilance, poison control centers, posology, selection of medicines, drug utilization review, and evaluation were being partially offered in healthcare institutes. Eight out of 13 respondents stated that drug use studies and pharmaco-economics were uncommon practices in healthcare settings in Pakistan. As an overview, the theme related to the current scenario of the pharmacy services being offered in Pakistan, along with subthemes, categories, and exemplar quotations, is provided in Numerous advantages for the provision of pharmacy services were indicated by participants. Better quality of life, improved adherence to therapy, and reduced drug-related problems (DRPs) were the patient-related benefits that emerged in discussion about the implementation of pharmacy services in Pakistan. Similarly, reduced burden on healthcare professionals and financial gains were the healthcare-system-related benefits explained by the study participants. As an overview, the theme related to benefits of implementing pharmacy services in Pakistan, along with categories and exemplar quotations, is provided in The study participants indicated that having a limited number of pharmacists, inadequate number and type of roles and training opportunities for pharmacists (8 out of 13 respondents), lack of patient knowledge about pharmacists and their role in healthcare delivery (6 out of 13 respondents), and suboptimal recognition by the government (8 out of 13 respondents) of pharmacists were the major barriers hindering the implementation of pharmacy services at all healthcare levels in Pakistan. Moreover, renting out pharmacist licenses (8 out of 13 respondents) and negative perceptions from doctors about pharmacists (7 out of 13 respondents) were the other barriers to successful implementation of pharmacy services at community and hospital healthcare levels, respectively. The theme related to barriers in implementation of pharmacy services, along with subthemes, categories, and exemplar quotations, is presented in The participants elaborated on various strategies for the improvement of pharmacy service provision in Pakistan. Most of the study participants emphasized the need to provide training opportunities for pharmacists (11 out of 13 respondents). Just under half (n = 6) of the pharmacists stated that pharmacy services could be improved by educating patients about the benefits of these services and by promoting the role of pharmacists in the healthcare sector, especially in community settings. At the hospital level, better interactions between doctors and pharmacists was deemed crucial by the study participants to improve the delivery of pharmacy services. As an overview, the theme related to strategies to improve pharmacy services in Pakistan, along with subthemes, categories, and exemplar quotations, is presented in In Pakistan, pharmacists have added value to the healthcare system as either manufacturers, purveyors, and dispensers of medicinal products, or as clinical service providers . NotwithThe emergent findings suggest that most of the pharmacist participants were familiar with the concept of “pharmacy services”. Most of the respondents also had a sound understanding of traditional pharmacy services . Similar to this study, the findings of studies from Middle-Eastern countries, such as Jordan, Iran, and Lebanon, indicate that most of the participants understand the concept of basic pharmacy services ,38. As fWhen asked about the services currently being provided in healthcare settings, the participants in this study viewed pharmacy services to be in their infancy in Pakistan. They further elaborated that conventional pharmacy services, such as dispensing, storage, sale, procurement, and distribution, were offered at the government, private, and community healthcare levels, whereas counselling, posology, selection of medicines, prescription monitoring, pharmacovigilance, and drug utilization review were partially offered by the pharmacist at these settings. Contrary to this, pharmacists in Saudi Arabia play promising roles, not only in providing pharmacy services such as dispensing, procurement, and storage, but they are also engaged in dose adjustment, posology, pharmacovigilance, and prescription monitoring to ensure the safe and affordable use of medicines . PatientMultiple advantages of providing pharmacy services were highlighted, which included benefits related to both patients and the healthcare system. Participants were largely of the opinion that the implementation of pharmacy services in Pakistan could lead to improved quality of life for patients, reduced DRPs, rational use of medicines, and financial gains. Likewise, a study conducted in the UK affirmed that pharmacy services contribute towards sound patient care, positive treatment outcomes, and financial and humanistic benefits .Upon asking the respondents about the obstacles that impede the progression of pharmacy services in Pakistan, they asserted that most of the shortcomings across all sectors and levels within healthcare were due to regulatory processes. The major barriers were suboptimal enforcement of rules and regulations, limited recruitment of pharmacists and consequent insufficient pharmacy workforce, and lack of training opportunities. Similarly, studies conducted in Ethiopia and Malaysia showed that the shortage of pharmacists and insufficient jobs for pharmacists were the major barriers to the implementation of pharmacy services, and these legislative changes were required for proper implementation ,47. EvenTo overcome the aforementioned barriers, a number of strategies are proposed from our study. The most important way of helping the situation is to train Pakistani pharmacists well and to equip them with the professional skills required to offer enhanced cognitive services. As evidence of this, a study conducted in Brazil also reported that clinical training and resulting improved knowledge of pharmacists favored the implementation of pharmacy services . AdditioThis study contributes to a sparse literature around perceptions of the scope of implementation of pharmacy services in Pakistan. However, there are limitations to this study. Firstly, the study participants were all from a single university in one city in Pakistan. As such, the findings cannot be generalized  across tThe participants in this study had an understanding of the terms related to “pharmacy services”. Participants suggested that traditional pharmacy services, such as dispensing and procurement of medicines, were offered across various healthcare settings. Meanwhile, counselling, prescription monitoring, pharmacovigilance, poison control centers, drug utilization review, pharmaco-economics, and pharmaco-epidemiology are suboptimal in Pakistan. The pharmacists considered the reduction of medication-related problems and financial benefits to the healthcare system as possible benefits derived from pharmacy service provision. This study has identified strategies to improve pharmacy services, including campaigns for greater awareness of patients about pharmacy services, proper implementation of the DRAP Act 2012 rules and regulations, better collaboration amongst healthcare professionals, and more robust training opportunities for pharmacists. A number of barriers that have hindered the successful implementation of pharmacy services in Pakistan were identified. These involve various healthcare disciplines in Pakistan, including pharmacists themselves, physicians, and patient-related barriers. Future research should focus on the viewpoint of health policy-makers in Pakistan.Pharmacy services in Pakistan are not fully implemented at all levels of healthcare, including public and private sector hospitals or within community drug retail outlets. The findings of this study are expected to assist policy-makers, especially those within the Pharmacy Services Division of DRAP, to take multifaceted corrective measures to overcome barriers preventing successful implementation of pharmacy services in Pakistan. This study outlines the current scenario of pharmacy services in Pakistan and provides strategies to improve enhanced cognitive pharmacy service provision in this nation. A national survey of pharmacists could verify the generalizability of these findings. It is recommended that initially policy-makers implement these strategies at a pilot scale and later scale this up upon achieving successful results. This study serves as a basis for future in-depth studies to explore the views of the wider healthcare team and policy-makers involved in developing pharmacy services policy in Pakistan.


Pesticides are among the most important contaminants in food, leading to important global health problems. While conventional techniques such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS) have traditionally been utilized for the detection of such food contaminants, they are relatively expensive, time-consuming and labor intensive, limiting their use for point-of-care (POC) applications. Electrochemical (bio)sensors are emerging devices meeting such expectations, since they represent reliable, simple, cheap, portable, selective and easy to use analytical tools that can be used outside the laboratories by non-specialized personnel. Screen-printed electrodes (SPEs) stand out from the variety of transducers used in electrochemical (bio)sensing because of their small size, high integration, low cost and ability to measure in few microliters of sample. In this context, in this review article, we summarize and discuss about the use of SPEs as analytical tools in the development of (bio)sensors for pesticides of interest for food control. Finally, aspects related to the analytical performance of the developed (bio)sensors together with prospects for future improvements are discussed. Pesticides are among the most used products in the agri-food industry for the control, prevention and elimination of pests. According to the target pest, they can be classified in insecticides, acaricides, fungicides, bactericides, herbicides, etc. The main pesticides are made of carbamates, dinitrocompounds, organochlorines, organophosphates, pyrethroids, neonicotinoids or triazines, among others . HoweverThe United Nations (UN) estimates that 200,000 deaths from acute poisoning occur each year due to pesticides, 99% belonging to developing countries . ContinuThe official methods for the determination of pesticides are based on chromatography, such as High Performance Liquid Chromatography (HPLC), HPLC-MS/MS and Gas Chromatography coupled to Mass Spectrometry (GC-MS/MS) ,11,12,13However, the use of traditional electrodes requires relatively high sample volumes and quite complicated cell set-up, as they are not suitable for “in field” applications. In this sense, screen-printed electrodes (SPEs) have emerged as outstanding alternatives, overcoming the limitations of traditional electrodes. Screen printing is a well-developed technology widely used since the 1990s for the mass production of disposable and economical electrochemical sensors. This production process is carried out in several stages, as illustrated in Screen-printed electrodes (SPEs) are manufactured on ceramic or plastic substrates, in which different types of inks  are printed. In addition, these inks can be modified with nanomaterials or enzymes among other compounds, improving the analytical characteristics of the (bio)sensors developed from such electrodes.SPEs satisfy the need for highly reproducible, sensitive and cost-effective detection methods, with additional advantages related to the low cost of production, flexibility in design, small size and ease of electrode surface modification ,21,22,23In this review, recent applications of SPEs for the electrochemical detection of pesticides are summarized, giving a critical vision on the advantages, drawbacks and perspectives.The typical electrochemical techniques used for pesticides detection, after following one of the (bio)sensing routes schematized in Cyclic voltammetry (CV) is used to study the different electrochemical processes that take place when applying a potential scan. The measured peaks of current provide information on the oxidation and reduction processes of an electroactive specie. In addition, it provides information on the type of process object of study: (i) reversible; (ii) irreversible or (iii) quasi-reversible, depending on the separation between the anodic and cathodic peaks. Another characteristic that popularizes the CV is its ability to give information about the nature of a process in terms of adsorption and diffusion characteristics.Differential pulse voltammetry (DPV) technique consists in applying a sequence of pulses of constant amplitude superimposed on a stepped potential increase. The current intensity is measured just before applying the pulse and at the end of it. The response obtained is the difference between the two current intensities, in relation to the potential at the start of the pulse, giving rise to a peak-like response.This technique is used to determine oxidation or reduction processes depending on the analyte concentration. In general terms, DPV has better sensitivity than cyclic voltammetry.In square wave voltammetry (SWV) technique, a large amplitude square wave potential sweep is applied with a stepped potential ramp. Current intensity is measured at the end of each applied pulse in the potential sweep cycle.Generally, SWV is more sensitive, faster and more selective than DPV, since the background current is minimized.Chronoamperometry (AC) is a determination technique where a constant current intensity is applied for a certain time. During to the application of such potential, the electroactive analytes present in the solution are oxidized or reduced, generating and associated current, proportional to the amount of analyte. The main advantage of this technique compared with voltammetries is related to its simplicity.Electrochemical impedance spectroscopy (EIS) is a technique commonly used to evaluate parameters of charge transfer, corrosion processes, double-layer formation, or modification processes of the electrode surface.The EIS is given by the Nyquist diagram where values of the load transfer resistance and the resistance of the solution are obtained. Depending on the semicircle obtained in the Nyquist diagram, it can be determined if there is an impediment to the charge transfer or if it is favored by the modification of the working electrode. This technique is widely used in label-free biosensing formats, where the change in the impedance upon the analyte biorecognition allows its determination.Enzymatic sensors are the most widely used for the determination of pesticides on SPEs. Two are the main detection routes based on enzymes: (i) enzymatic inhibition route, where the pesticide inactivates the enzyme and (ii) catalytic route, where the pesticide is hydrolyzed by the enzyme generating an electroactive compound ,103. As stated above, inhibition reactions make enzymes inactive in the presence of pesticides. This is the case of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase (Tyr) and alkaline phosphatase (ALP).AChE is one of the most commonly used enzymes for the determination of organophosphorus pesticides (OP). The reaction catalyzed by AChE is the following:The presence of OP results in the inhibition of the enzymatic activity due to phosphorylation of the serine residue of the active center of the enzyme, which blocks the hydrolysis of acetylcholine (ACh). Therefore, the higher the concentration of OP, the more blocked the active center will be and the lower the signal of oxidation or reduction of the enzymatic products. Detection based on other enzymes relies in the same principles.As shown in However, in most cases working electrodes are modified with different materials so as to improve the efficiency of the enzyme immobilization and thus the pesticide analysis. An example of simple electrode modification is the reported by Arduini et al.  where Pr3O4) coupled to a graphene (GR) film on a SPCE 3-/4- ions. Under the optimal conditions LoDs ranging from of 0.003 to 0.3 nM were obtained for Profenofos, Phorate, Isocarbophos and Omethoate. The aptasensor was also successfully applied for rapeseed and spinach samples, after an extraction treatment.Aptamers have also been immobilized on SPE in composites with reduced graphene oxide (rGO) and Cu nanoparticles (CuNP). That is the case of the work reported by Fu et al.  for the AuNP-modified SPEs were also proposed for the immobilization of an aptamer specific for Diazinon (DZN) . IncreasCells and microorganisms can also be used as recognition elements in biosensors. In this case the analytical signal is commonly related to the activation or inactivation of cellular respiration upon analyte interaction, leading to the production of electroactive metabolites. A representative approach consists in the genetic modification of microorganisms with enzymes, such as OPH for the determination of organophosphorus pesticides by measuring the enzymatically produced p-nitrophenol ,197,198.Spinacia oleracea, Senecio vulgaris and its atrazine resistant mutant immobilized with BSA-GA on the SPE for the detection of herbicides that selectively block the electronic transfer activity of PS II biomediators  are emerging platforms with outstanding potential for their use as transducers in electrochemical (bio)sensing of pesticides. Their well-known advantages in terms of disposability, portability and low-volumes required, among others, make them ideal for the “in field” detection of pesticides at the point of need. Moreover, their versatility and easy modification with different materials is of key relevance for reaching ultralow detection limits that allow to detect the pesticides at the maximum levels allowed by the legislation. In this line, the use of nanomaterials, such as carbon-related ones  and metallic nanoparticles is the object of an extensive research in recent years.The (bio)sensing strategy to be followed for the pesticide detection must be carefully studied for each concrete case.The simpler and faster strategy consists in the direct detection, taking advantage of the electroactivity of some pesticides, that is the presence of functional groups with red-ox properties. However, a limited group of pesticides can be sensitively detected through this route, typically at levels of µM–nM. Enzymatic sensors are the most widely used, benefitting from the wide range of pesticides able to be detected and the high sensitivity reached, typically at levels of nM–pM. However, the use of enzymes entails important limitations related to their low stability over time and their sensitivity to changes in temperature and pH, among others, so alternative biosensing methods based on antibody receptors, are becoming popular in the last years.The main advantages of such immunosensors over the enzyme-based ones rely on the higher stability of antibodies together with their superior selectivity and specificity. The detection limits reached are quite similar to the ones obtained through the enzymatic route, typically at pM levels. The high cost and low availability of monoclonal antibodies and the small size of the pesticides that usually avoids sandwich-based approaches are important limitations that should be considered before selecting this biosensing approach. In conclusion, the selectivity and sensitivity/detection limit required, and the availability of specific enzymes and antibodies are the main parameters that should define the detection strategy more suitable for each particular application.As far as we know, none of the reported electrochemical (bio)sensors for pesticides detection on SPEs are commercially available yet. In this line, some important issues should be solved for the implantation of such (bio)sensing systems for routine analysis, as alternative to centralized laboratory-based methods . Efficiency and long-term stability of the enzymes and the antibodies are crucial issues that are not addressed in most of the reviewed works. Moreover, multi-detection abilities should be strongly required for real applications in pesticides screening. Efforts in this sense should be the next at the current state of the art.Overall, the higher potentiality of SPE-based pesticide (bio)sensors is in the decentralized “in field” analysis, in our opinion. The combination of such miniaturized electrochemical transducers, the cheap and portable electrochemical instruments and the stability of mainly the antibodies make altogether ideal for such applications. In the case of the immunosensors, the most challenging issue is related to the sampling, washing, etc. steps required, which limits their use by non-skilled people and consequently their commercial implantation. The combination with microfluidics seems to be of key relevance here, so high efforts in this sense are previewed for the coming years.