Interdisciplinary combinations of medicine and engineering are part of the strategic plan of many universities aiming to be world-class institutions. One area in which these interactions have been prominent is rehabilitation of stroke hemiplegia. This article reviews advances in the last five years of stroke hemiplegia rehabilitation via interdisciplinary combination of medicine and engineering. Examples of these technologies include VR, RT, mHealth, BCI, tDCS, rTMS, and TCM rehabilitation. In this article, we will summarize the latest research in these areas and discuss the advantages and disadvantages of each to examine the frontiers of interdisciplinary medicine and engineering advances. Stroke is a serious cerebrovascular disease characterized by sudden and acute onset and rapid neurological deficits, which is the world's leading cause of disability and the second leading cause of death , leavingVirtual reality (VR) combines the VR technology characteristics of autonomy, interactivity, and presence with rehabilitation training, to provide novel methods for stroke patients to undergo neurorehabilitation in virtual environments . VR is eA study of 10 stroke patients found that, by wearing a head-mounted display, immersive VR mirror therapy could treat poststroke upper limb paresis . StudiesSome studies have suggested that VR has more significant effects than conventional therapy in improvement of gait speed, stride frequency, and step length , and canThough promising, VR technology does have room for further development as VR can induce eye fatigue and physical fatigue during treatment, commonly manifesting as motion sickness. Motion sickness occurs when the screen display and the user's visual response are delayed . Some stRehabilitation robotics have expanded in recent years as the result of extensive communication and interaction between clinicians and engineers, leading to the development of new technologies that stimulate . RehabilOne study included patients with subacute stroke that were given three weeks of intensive robot training. At the conclusion of the study, their athletic and living ability significantly improved . AnotherThe current paradigm is that many repetitions of rehabilitation actions are effective for promoting recovery. However, traditional rehabilitation therapists have limited energy and the number of repetitions they can manually perform is too small to induce neuroplasticity. Thus, robot-based rehabilitation training may lead to higher exercise repetition intensity, which could promote neuroplasticity. However, some robots, such as Robot-Assisted Step Training (RAST) , provideSome recent research has also studied the portability and comfort of robots. For example, a biofeedback wearable robot based on human-computer interaction, compared to EMG feedback, can better improve patient compliance and can help accelerate the recovery of ankle-foot deformity after stroke . AnotherRehabilitation of stroke hemiplegia is a long process, and the rehabilitation clinic resources are limited. When discharged from the hospital, hemiplegic patients are usually provided with a written family exercise plan to guide their recovery in the chronic stage of stroke. However, these plans rely on the patient's consciousness, and are unsupervised, which can limit efficacy. To address this, mobile health  can provide remote monitoring and remote consultation . mHealthOne mHealth example is smart shoe technology based on the Personalized Self-Management Rehabilitation System (PSMrS), which monitors the patient's movement through inductive insoles and projects sensor data on screen to provide feedback to the patient that can be assessed and monitored at home . AnotherBrain-computer interface (BCI) is a direct connection path established between the human brain and external devices. This technology translates the neurophysiological signals in the brain into control signals that can operate external devices or computers to assist in performing different tasks . The accMany clinical studies have shown that BCI training is effective in the rehabilitation of hemiplegia after stroke –45. The BCI via wearable and wireless EEG headsets can record EEG signals in different environments, making EEG-BCI more flexible, yet the recording quality of current headset technology usually declines after about an hour , 51. DryTranscranial direct current stimulation (tDCS) is a form of noninvasive brain stimulation (NIBS). Under physiological conditions, competition between cerebral hemispheres maintains the balance of bilateral cortical excitability. After a stroke, the balance between the hemispheres is disrupted, the excitability of the affected hemisphere decreases, and the excitability of the unaffected hemisphere increases . In tDCStDCS is widely used clinically and has significant effects on the recovery of gait speed and gait quality . StudiesThe study found that stimulation showed more significant changes in interval stimulation after stroke  compared to daily tDCS. The results showed that the density of cortical dendritic spines increased significantly and the expression of pannexin-1 mRNA involved in hypoxia depolarization decreased . CurrentrTMS is another type of noninvasive brain stimulation (NIBS), which modulates the excitability of neurons by passing current through an insulated coil. The adjustment of excitability depends on the rTMS parameters. High-frequency stimulation can have excitatory effects within the stroke-damaged hemisphere, and low-frequency stimulation with inhibitory effects is used for the undamaged hemisphere . High-frrTMS can delay or prevent the death of hippocampal neurons in adult rats with cerebral ischemia. The specific preservation of neurons depends on the stimulation mode and the time interval between ischemia and stimulation. Maximum neuronal protection can be achieved by applying high-frequency rTMS (at least 128 seconds) in the first 48 hours of ischemia occurring . IschemiAlthough rTMS has been widely used to improve upper limb movement in stroke patients with hemiplegia, a systematic review and meta-analysis of 199 patients showed that current literature is insufficient to support the conclusion that rTMS combined with upper limb training is more effective than upper limb training alone . The 200Theta burst stimulation (TBS) is a newly developed form of rTMS that mimics the firing patterns of hippocampal pyramidal cells during wakefulness in rodents exposed to new environments, producing low-intensity bursts of stimulation to coordinate cortical excitability . IntermiTraditional Chinese medicine (TCM) is a treasure of the Chinese nation and when combined with modern science and technology can result in truly optimal integrations of medicine and engineering to advance TCM rehabilitation technology. TCM treatment of stroke hemiplegia typically includes acupuncture, massage, and rehabilitation training, supplemented by drug, psychological, physical, and exercise therapy, to promote the recovery of limb function. TCM first began taking advantage of advances in medicine and engineering through combination of acupuncture and electricity in the 1950s when Zhu Longyu established electroacupuncture therapy . AcupuncWith the rapid development of science and technology, artificial intelligence, brain-computer interface, and other technologies have been widely used in clinical practice. Interdisciplinary combination of medicine and engineering is an inevitable trend in the development of modern medicine. However, as summarized above, there are some areas to be improved regarding appropriate patient selection and technical optimization. A major problem that exists is that most of the current interdisciplinary combination of medicine and engineering methods cannot prove that they are better than conventional therapy when used alone, and require further technical optimization. Additionally, in some studies, patients in the subacute phase after stroke were included in the comparison of treatment methods, but these data were not convincing because spontaneous recovery in the subacute phase would interfere with experimental results. There are also unique differences among stroke patients with hemiplegia, such as those suffering from ischemic or hemorrhagic stroke or those in the subacute or recovery phases. A limitation of some studies is that they do not specify the category of patients in the study. Many interdisciplinary combinations of medicine and engineering treatment methods also lack standardized programs for intervention measures, stimulation parameters, and treatment course standards, which likely contribute to a lack of mechanistic insights regarding these methods. The interdisciplinary combination of medicine and engineering in the field of TCM rehabilitation has great potential, but the foundation is relatively weak, and further development and research are needed.ε2/ε4 of apolipoprotein E (APOE) is strongly associated with recurrence of OAT-ICH. Biffi et al. [ε2/ε4 variants to predict OAT-ICH recurrence. Liew et al. [In addition to helping doctors treat patients and promote their recovery, interdisciplinary combination of medicine and engineering has also been initially used to predict the incidence and prognosis of stroke hemiplegia. Chen and Song  establisi et al.  used neuw et al.  also useInterdisciplinary combination of medicine and engineering refers to integration and collaborative innovation of medicine and engineering centering on existing medical needs. This allows the most advanced technological means of engineering to help solve clinical needs, and aid doctors in quick and accurate diagnoses, which promotes rapid patient recovery . HoweverTo break through the bottleneck of development, in addition to the special support of policies and funds, it is suggested to promote the interdisciplinary research of medicine and biology, physics, material science, computer science, and other disciplines, to form many cross-disciplines with the characteristics of interdisciplinary combination of medicine and engineering. However, the most important thing for cultivating high-level interdisciplinary combination of medicine and engineering talent is the promotion cross-disciplinary thinking and innovation for trainees who are proficient in medical and engineering knowledge and can actively find problems encountered in clinical practice and solve them with engineering methods.


Acquired reactive perforating collagenosis is a rare skin disorder characterized by the presence of umbilicated pruritic papules and nodules. Transepidermal elimination of altered and perforating bundles of basophilic collagen from the epidermis is a characteristic histologic feature of acquired reactive perforating collagenosis. Along with its well-known association with systemic diseases such as diabetes mellitus, chronic renal failure, and dermatomyositis, there are reports of acquired reactive perforating collagenosis being associated with malignancies. Herein, we present a case of acquired reactive perforating collagenosis associated with chronic lymphocytic leukemia, prostate adenocarcinoma, and Graves's disease. Clinicians are required to be more vigilant in evaluating patients with acquired reactive perforating collagenosis due to its unique association with malignancies and other systemic diseases. Acquired reactive perforating collagenosis (ARPC) is a rare skin disorder with characteristic clinical and histological features and a typical adult-onset. ARPC is one of the four diseases that belong to the disease group called primary perforating dermatosis. Patients commonly present with characteristic umbilicated necrotic papules or nodules having a localized or generalized distribution. Histopathological examination of these lesions exhibits transepidermal elimination of altered and perforating bundles of basophilic collagen from the epidermis.A 75-year-old male was admitted to our dermatology department with a widespread, itchy rash that had developed during five months. The patient did not respond well to oral antihistamine, topical corticosteroid cream, and narrowband ultraviolet B phototherapy (UVB) for 13 sessions. He was diagnosed with chronic lymphocytic leukemia (CLL) and Graves’ disease (GD) 11 and 12 years ago, respectively. He was on drug-free follow-up for CLL and GD at the time of hospital admission. The patient was otherwise healthy with no family history of diseases.Dermatological examination revealed multiple, widespread, dome-shaped umbilicated papules and nodules with an average diameter of ∼4 mm distributed on the trunk, face, and upper and lower extremities . Some ofLaboratory results of complete hemogram, blood sugar, liver, and kidney function tests were found to be within normal limits. Histopathological evaluation of a representative skin lesion taken from the left leg showed acanthosis, basket weave orthokeratosis, increased number of vessels in the superficial dermis, and perivascular and interstitial inflammatory infiltrate with erythrocyte extravasation. Punched-out ulceration with cellular debris was observed in the crateriform invagination and transepidermal elimination of collagen fibers . ARPC waReactive perforating collagenosis (RPC) can be both hereditary  and acquired.Transepidermal elimination of altered dermal collagen is thought to be crucial for the pathogenesis of ARPC. However, the etiology and pathogenesis of ARPC are not fully understood. Tsuboi et al. suggested that systemic diseases contribute to the degeneration of collagen fibers and the production of altered dermal products either directly or indirectly.ARPC is commonly associated with systemic diseases such as DM, chronic renal failure, and dermatomyositis. Review of the literature revealed 14 published cases of ARPC that were associated with malignancies. Despite the small number of these cases, some authors suggest to consider ARPC as a paraneoplastic syndrome.In ARPC, lesions are usually widespread and may appear anywhere on the body with a slight predilection for the lower extremitiesA standardized treatment protocol for ARPC does not exist and treatment remains challenging. Controlling pruritus and the underlying extracutaneous disorder is important for the management of ARPC. In drug-induced cases, cessation of the suspected drug is crucial. Topical corticosteroids, oral antihistamines, emollients, UVB phototherapy, systemic retinoids, allopurinol, and cryotherapy are used for the treatment of ARPC.As far as we know, there is no report on ARPC being associated with CLL and GD; however, a case of ARPC associated with prostate carcinoma has been reported before. Herein, we reported a case of ARPC associated with CLL, prostate adenocarcinoma, and GD mimicking scabies, folliculitis, or prurigo simplex. We emphasize the importance of the association of ARPC with malignancies. Clinicians are required to be more vigilant in evaluating patients with ARPC considering its unique association with malignancies as well as other systemic diseases.None declared.Leyla Huseynova: Approval of the final version of the manuscript; conception and planning of the study; elaboration and writing of the manuscript; obtaining, analysis, and interpretation of the data; effective participation in research orientation; intellectual participation in the propaedeutic and/or therapeutic conduct of the studied cases; critical review of the literature; critical review of the manuscript.Neslihan Akdogan: Approval of the final version of the manuscript; conception and planning of the study; elaboration and writing of the manuscript; obtaining, analysis, and interpretation of the data; effective participation in research orientation; critical review of the literature; critical review of the manuscript.Özay Gököz: Approval of the final version of the manuscript; elaboration and writing of the manuscript; obtaining, analysis, and interpretation of the data; effective participation in research orientation; critical review of the literature; critical review of the manuscript.Sibel Ersoy Evans: Approval of the final version of the manuscript; elaboration and writing of the manuscript; obtaining, analysis, and interpretation of the data; effective participation in research orientation; intellectual participation in the propaedeutic and/or therapeutic conduct of the studied cases; critical review of the literature; critical review of the manuscript.None declared.


The ongoing pandemic of COVID-19, caused by the severe acute respiratory syndrome coronavirus SARS-CoV-2), has dramatically shown how the elderly are fragile in spite of advancement in healthcare and medical attention in Western countries  sleep and rapid-eye-movement (REM) sleep characterizes the organization of human sleep. Current evidence confirms the presence of age-related changes in sleep duration and architecture, so that older adults could have a reduced ability to sleep rather than a reduced sleep need . DecreasInflammation is a physiological response of the immune system to infection and injury regulated by different immune cells and inflammatory factors. Inflammatory molecules  act locally but also at a distance. Locally, they take action on nearby immune cells to stimulate their recruitment/activation, and on the vascular system to induce vasodilation and vascular permeability. They also communicate over great distances to other organs, establishing a systemic inflammation that can involve brain inflammation, also known as neuroinflammation . During With aging, the circadian indicators of the sleep-wake organization, i.e., sleep onset, body core temperature, motor activity, and melatonin phase, have an earlier daytime emergence ; the cirOne of the most consistent evidence of age-associated circadian disruption is the age-associated changes in sleep, with its quantity progressively reducing (sleep deprivation) and quality worsening from around 55 yrs. of age to become worst over 65 yrs. also in subjects without major sleep disorders. Major sleep disorders and especially sleep deprivation are associated to immune dysfunction and chronic inflammation and to higher risks of infection and psychiatric, cerebral/cardiovascular, metabolic/hormonal co-morbidity and related mortality irrespective of age, according to surveys and population-based studies , 22. ConThe circadian system and sleep have emerged as important intertwined regulators of immune defense , 24. TraAdditionally, adequate sleep quantity and quality support the immune function, reduce infectious risks and improve vaccination responses by regulating immunological memory and humoral immunity . On the The spatiotemporal regulation of gene expression and associated functions contribute to maintain physiology and behavior.From accumulating evidence new therapeutic opportunities have been generated for two main reasons: first, by leveraging rhythmic aspects of some diseases in occurrence, including cardiovascular or neurological ones  and infeThe relationship between aging and circadian disruption is bidirectional, with aging associated with circadian dysfunction, and behavior and genetic disruption of the circadian clock leading to aging-like phenotypes. The result is the establishing of circular, self-sustaining mechanisms of dysregulation with potentially severe impact on health. The bidirectional relationship among sleep/circadian rhythm and immunity has received minor attention to date mainly if we consider the emergence of sleep disorders in the adulthood that persist until the elderly. It remains unclear the extent to which sleep disorders affect the immunosenescence in the later life. During the lifetime, a progressive cumulative decline in physiological homeostasis occurs, thus reducing the quality of life, and promoting isolation and adverse health outcome in the elderly . In thisThe link among the disorders of circadian clock, sleep and immunosenescence qualify today as a high-priority healthcare, welfare and social constraints with high estimated direct and indirect costs, that hover higher in the elderly  if countThe circadian pattern of the immune system contributes to shape the host ability to respond to viral infections and the host–pathogen interaction. Viral infections and other infectious diseases influence the host circadian clock drive. Therefore, in the complex host–pathogen interaction a role of timing can be suggested, offering new therapeutic opportunities based on chrono-modulated antiviral strategies. A more in-depth research in the interrelationship of circadian system and immunity in the elderly, with multidisciplinary competences, from (chrono)biology, (chrono)pharmacology, neuroimmunology, sleep to integrative physiology, is advisable and possibly overdue.SG and WS conceived the manuscript. SG, EG, and PL wrote the manuscript. NB and WS revised the manuscript. All authors contributed to the article and approved the submitted version.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.


Intraorbital metallic foreign bodies have varied clinical presentations. Here, we report the unusual case of intraoperative evidence of spontaneously healed posterior scleral perforation in a severe ballistic trauma without previous instrumental signs of penetrating wound and complete visual restoration after surgery.The patient was hit by several lead hunting pellets in the chest, abdomen, limbs, face and orbit. Computed Tomography (CT) images revealed the presence of a pellet within the orbitary cavity, close to the optic nerve, with no signs of penetrating ocular wound. While performing vitrectomy for severe vitreous hemorrhage, a point of strong adherence between a old hemorrhage and retinal surface was identified and managed conservatively, as it was attributed to trauma related-impact area. So, lead foreign body took an unusual trajectory impacting the globe and finally lodging back in the deep orbitary cavity, in absence of significant ocular injury and with visual prognosis preservation.Our findings provide further information on orbital injuries from airguns, a theme of growing popularity and concern. Intraoperative recognition of hardly removable old hemorrhagic clot as self-blockage site of posterior scleral penetrating trauma, allowed for surgical stabilization and minimal solicitation of the area to avoid inadvertent perforation. In the recent past, like-gun shot wounds have become a growing clinical concern due to the increasing popularity of airguns, with advances in compressed-gas technology leading to a significant increase in power and velocity of these weapons , 2.Common post-traumatic orbital injuries include anterior chamber injuries, injuries to the lens, open-globe injuries, ocular detachments, intraorbital foreign bodies, carotid cavernous fistula, and optic nerve injuries , 4. MoreA 58-year-old man, victim of airgun aggression, presented to our General Emergency Unit of the Hospital Umberto I of Rome, ‘Sapienza’ University with multiple wounds such as entry holes in the subcutaneous space and in muscles of thorax, abdomen, limbs, groin and face Fig. .Fig. 1CHe underwent total body CT and no vital organs resulted damaged. Blood tests were within normal limits and the patient was fully conscious, well oriented, without signs of urgent surgical need. As far as his orbital involvement, it was described full thickness wound in his right upper eyelid, swelling and ecchymosis in his right orbital region, accompanied with important vision decline consequently to massive vitreous hemorrhage in the right eye. CT scans of the orbital region revealed the presence of a hunting lead pellet within the orbitary cavity, supero-temporally close to the optic nerve, in the deep retrobulbar space between the optic nerve and the lateral rectus muscle, with no penetrating wound of the globe Fig. . The patAdditional file 1: Video. 25 G Pars-plana Vitrectomy and IOL implantation. Intraoperative identification of the ocular site of impact and related management.The orbitary cavity is vulnerable to injury from airgun pellets and direct hits invariably result in severe structural and functional damage to the eye or other orbital contents. Foreign bodies can reach the orbital space because of bulbar double perforation or via lid lacerations through peribulbar tissues. In the latter way, as the globe integrity is maintained, visual acuity is preserved –8.Here we report the intraoperative identification of a fibrinous mass, acting as plug, resulting hard to be removed by the use of vitrectome, and recognized as the ocular point of impact of an intraorbital lead pellet. On the basis of clinical features and radiological appearance of the foreign body, we speculate that it traversed the orbital septum through the right upper lid and peribulbar soft tissues, and superonasally impacted the eye without being able to penetrate it. The airgun pellet then definitely localized itself at the bottom of the orbitary cavity, superotemporally to the orbital apex and medially to the lateral rectus muscle. This lucky trajectory preserved the eye, the optic nerve and the whole orbitary content from severe and irreversible structural and functional damage. The massive vitreous hemorrhage and lens subluxation were surgically managed, and the fibrin mass was maximally reduced, but not further solicited, in order to prevent any risk of unintentional perforation. We decided not to remove the intraorbital pellet as it was not readily accessible, and the patient did not complain any orbital complications such as orbital cellulitis or systemic toxicity. About that, most metals retained in the orbit, including lead, are inert and in the absence of infection cause no disturbance and should be managed conservatively in the absence of specific indications for removal , 10.We immediately judged this case as unique for the clinical scenario and surgical management. A few articles in Literature are based on this topic, especially when the pellet is located posterior in the orbit and injury occurs without permanent visual impairment –13.We believe in the importance of disclosing the information contained in this report and relative multimedial material, in order to promote exact recognition of ocular trauma point in case of a fibrin plug resulting hardly removable using vitrectome, and so to avoid any potential induced perforation. The combination of documented intraoperative evidence of ocular site of impact, absence of any instrumental data of penetrating wound and total visual recovery, represented an exceptional occurrence and no similar cases have been published in Literature hitherto.


In vitro direct reprogramming approaches primarily aim to model the pathophysiological development of neurological disease and identify therapeutic targets, while in vivo direct reprogramming aims to develop treatments for various neurological disorders, including cerebral injury and cancer. In both approaches, there is progress toward developing increased control of subtype-specific production of induced neurons. A majority of research primarily utilizes fibroblasts as the donor cells. However, there are a variety of other somatic cell types that have demonstrated the potential for reprogramming into induced neurons. This review highlights studies that utilize non-fibroblastic cell sources for reprogramming, such as astrocytes, olfactory ensheathing cells, peripheral blood cells, Müller glia, and more. We will examine benefits and obstructions for translation into therapeutics or disease modeling, as well as efficiency of the conversion. A summary of donor cells, induced neuron types, and methods of induction is also provided.Direct cellular reprogramming exhibits distinct advantages over reprogramming from an induced pluripotent stem cell intermediate. These include a reduced risk of tumorigenesis and the likely preservation of epigenetic data.  Neurons are the primary functional unit of the brain and are a diverse, dynamic, and essential cell population of great importance to our study of cognitive function as well as therapeutic development for brain injury. Direct reprogramming of somatic cells to various subtypes of induced neurons (iN) has great potential in the field of neuroscience research. This approach allows for disease modeling in neurons of human origin (even from the same patient) as well as the possibility for developing regenerative medicine therapies. The interest in developing direct reprogramming techniques can be attributed to advantages that arise from technical differences between induced pluripotent stem cell (iPSC)-derived iN and directly reprogrammed iN derived from somatic cells. The direct reprogramming approach can utilize endogenous patient cells as donors, removing the need for the complicated and expensive process of iPSC derivation, reprogramming, and engraftment. Furthermore, it reduces the risk of tumorigenesis/teratogenesis due to the lack of a pluripotent intermediate state and holds the potential of preserving the epigenetic memory of the donor cell, which has a tremendous impact on the accuracy of disease modeling. Using an epigenetic biomarker of aging, DNA methylation, data showed that epigenetic age of the donor cell was consistent with the corresponding directly reprogrammed iN . It is rin vitro and in vivo. In vitro direct reprogramming methods have developed with the goal of more precise modeling of the pathophysiological development of disease and mapping potential therapeutic targets, while the in vivo direct reprogramming approach aims to treat various neurological disorders and injuries, by targeting the patient’s local endogenous cells for reprogramming  for resident striatal astrocytes to tyrosine hydroxylase-expressing iN , or NG2 glia, are a promising donor cell source because of their abundance and proliferative capacity . NG2 gliA recent study demonstrated the reprogramming potential of microglia into iN with NeuroD1 via LV at relatively high efficiency (25–35%) . Direct in vivo survival of these cells, neural integration of iN into the appropriate circuitry, functional analysis at the cellular and network level, and finally the assessment of potential deleterious effects of iN introduction. Addressing these issues will help drive the field forward. With correlation of electrophysiology, calcium imaging, and integration into neural circuitry, preclinical studies have made significant strides toward producing a fully realized, directly reprogrammed iN.Direct reprogramming has tremendous potential. In one study, directly reprogrammed neural precursor cells administered during the subacute phase of stroke demonstrated promising results for recovery in motor skills . AnotherKK completed a comprehensive review of the literature and produced initial drafts of the manuscript. MT provided draft review, edits, and corrections. DP provided content review and edits. RM supervised the activities of KK, produced edits and correction of drafts of the manuscript, and completed the final version of the text. All authors contributed to the article and approved the submitted version.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.


Oncorhynchus mykiss) production is based on all-female production with a high proportion of all-female triploid production in Europe. The main aim of this review is to present the recent knowledge regarding sex-reversed females (SRFs) of salmonid fish. We discuss the methods of sex reversal as well as their effects on the morphology and histology of the reproductive tract. We focus on the characteristics of SRF semen as well as the factors determining semen quality. The lower quality of SRF sperm compared to that of normal males has resulted in the need for the artificial maturation of semen. Most importantly, methods of semen storage—both short-term and long-term (cryopreservation)—that can improve hatchery operations are presented with the special emphasis on recent progress in development of efficient cryopreservation procedures and use of cryopreserved semen in hatchery practice. Moreover, we also address the emerging knowledge concerning the proteomic investigations of salmonid sperm, focusing primarily on the proteomic comparison of normal male and SRF testicular semen and presenting changes in SRF rainbow trout sperm proteome after in vitro incubation in artificial seminal plasma.Sex reversal has been used as a breeding strategy by salmonid fish to produce genetically and phenotypically single sex populations. Production of all-female fish has great importance for the creation of monosex female triploids of salmonid fish, which are valued for their sterility, lack of female maturation, and larger commercial size. Among salmonids, the majority of rainbow trout ( Sex-reversed females  are genetically females; however, owing to masculinisation, they are capable of producing spermatozoa . FemalesSalmo salar), has also been indicated [Oncorhynchus mykiss) production is based on all-female stocks, with a high proportion of all-female triploid fish occurring in Europe [The formation of female monoculture stocks of salmonids is of interest in commercial aquaculture because of its several advantages ,2,5,6. Indicated . The prondicated ,9. Nowadn Europe .To the best of our knowledge, the sex reversal of salmonid fish has previously been discussed by several excellent reviews ,11,12,13Oncorhynchus kisutch), and chinook salmon (Oncorhynchus tshawytscha) [Sexual development in gonochoristic teleosts is protracted and plastic, which makes it possible to reverse sex both toward the masculinisation of females and feminisation of males ,13,14. Twytscha) .There are two potential approaches for the production of monosex female groups of fish: Direct, based on the use of oestrogens to feminise genetic males  and indiIt has to be underlined that due to the commercial use of sex reversal, the detailed methods used to produce SRFs are proprietary to the producers and not available publicly, which restricts our knowledge (to an unknown extent) regarding the usefulness of specific protocols .There are a few major factors that need to be considered for the sex reversal of particular salmonid species: (i) The timing of sexual differentiation, (ii) methods of steroid administration and their dose, and (iii) species-specific characteristics important for the efficacy of sex reversal. The multi-factorial determination of sex reversal efficacy makes its control difficult and leads to different extents of male reproductive system development, such as sterile, bisexual, dysfunctional, and normal males  Table 1Table 1. The key steps for optimising the production of all-female populations are (a) determining the developmental stage at which gonadal differentiation occurs, (b) optimising the dose and duration of treatment with masculinising agents, and (c) distinguishing SRFs from normal males for breeding purposes.Salvelinus fontinalis), the pre-hatch period was found as a potential target period for sex reversal using androgen treatment [The timing of sexual differentiation during ontogenic development is critical for the advancement of the sex reversal procedure because the efficacy of steroids is at its highest while it is administrated during the so called “labile period”, i.e., starting when the gonads are undifferentiated and continuing through gonadal differentiation. Any deviations from the labile period decreases the efficacy of sex reversal and elevated doses of hormones and longer application times may be required to counteract these losses . Althougreatment . Moreovereatment . To compThere are three methods for steroid administration: (i) Injection , (ii) immersion in a static bath, and (iii) hormone supplementation in the diet. The injection of embryos or larvae is clearly not practical; consequently, the latter two methods are being adopted in hatchery practices, both separately and in combination . The selIn recent years, there has been growing concerns about the contamination of the aquatic environment with water pollutants due to anthropogenic activity, which is a serious threat for the reproduction of aquatic animals . EndocriThe effective steroid doses for sex inversion were found to vary both between and within salmonid species . SpeciesThe identification of SRFs is not necessary when genetic sex XX is clearly established either due to gynogenetic fish or the progeny of SRF and, in such a case, protocols aiming to produce functional  semen are desired ,44. The The consequences of hormonal treatments used to obtain SRFs are disorders of the anatomical development process of the gonads, which are manifested by the disruption of normal development, including the narrowing, disproportion, or atrophy of the gonadal lobes ,22,24,48Histologically, the testes of mature SRF rainbow trout consist of large lobules (about 0.5–1 mm in diameter) located in the centre and smaller lobules (about 50 μm) situated in the outer parts of the gonad . The thiThe masculinisation procedure significantly influences the gonadal development and sperm characteristics of fishes . SRFs ha9 spermatozoa mL−1  ,55,60,63Computer-assisted sperm analysis (CASA) is a tool that measures not only the sperm motility, but also sperm velocity parameters  and trajectory parameters . The vaSperm viability is a key determinant of sperm quality and is mainly based on the analysis of cell membrane integrity and functionality. Sperm viability is often linked to an intact plasma membrane, since the plasmalemma is pivotal for sperm interactions with other cells and their environment . It is nMitochondria, located in the sperm midpiece, produce a major part of the ATP essential for sperm motility ,75. Dama19 cells) and were significantly higher than those recorded for normal male rainbow trout . These differences likely reflect the lack of spermatic ducts in SRFs; the spermatic ducts are responsible for the synthesis and secretion of enzymes, monosaccharides, lipids, and proteins and regulate the ionic composition of the seminal fluid [The mineral constituents of seminal plasma have been identified as principal components affecting sperm quality. Their presence or absence interferes with osmolality, pH, and several spermatozoa functions, such as the motility activation of salmonid fish . The fival fluid ,83. Moreal fluid .−1) reflects the physiological osmolality of body fluids in fish [−1, similar to that of SRF rainbow trout, which are within the range of 290–342 mOsm kg−1  than in the seminal plasma of normal males (up to 450 U L−1) were recorded [−1 and are significantly higher in SRFs than in normal males (−1) were recorded [The antitrypsin activity (APA) and protein concentration are important determinants of fish semen quality. The APA of fish seminal plasma is involved in the regulation of spermatogenesis, protection of spermatozoa and reproductive tissues from proteolytic attack, and remodelling of connective tissue of the reproductive system ,89,90. Srecorded . High APrecorded , which arecorded ,89.−1 and were significantly higher than those in normal males  is a spermatozoal enzyme employed as an indicator of injuries that lead to the leakage of LDH from the spermatozoa into the seminal plasma . ConsequOxidative stress is a key factor in the pathophysiology of semen ,94. Oxid•), superoxide ion radical (O2•−), hydroxyl radical (OH•), and peroxyl radical (ROO•), as well as singlet oxygen (1O2). These molecules can interact with proteins, lipids, DNA, and RNA, promoting cell injury at several levels. Excessive ROS production leads to the peroxidation of polyunsaturated fatty acids, resulting in the production of aldehydes, such as 4-hydroxynonenal, acrolein, and malondialdehyde [−3 nmol mg−1 protein, significantly lower than those in the seminal plasma of normal males . Low le ± 1.2%; ). This sThe evaluation of antioxidant capacity of seminal plasma is mainly evaluated from the perspective of particular antioxidants. This strategy is time-consuming owing to the necessity of several compounds for analysis and the changeable levels of individual antioxidants . An alte−1 protein) and were not different from those recorded for normal males , glutathione reductase, methionine reductase, and superoxide dismutase have been identified . Catalas protein ).The quality of sperm DNA is an important indicator of the accurate transmission of genetic material from one generation to the next . OxidatiSRF sperm quality parameters are characterised by high variabilities . ,66. 64,6The viability of spermatozoa appears to be subjected to similar changes to the sperm motility, with a significant decrease after cryopreservation from about 90% to 60% . It shou+ from 3% to 7% in SRF rainbow trout sperm [The damages to spermatozoa during cryopreservation are—to a great extent—caused by oxidative stress, which arises as a result of the increased generation of ROS and/or a reduction in the number of accessible antioxidants . It has ut sperm . It shouut sperm . These aut sperm .The most important parameter for the evaluation of sperm quality in cryopreserved semen is the sperm fertilising ability. Similar to motility, the fertilising ability of cryopreserved SRF sperm is mostly lower than the values obtained using fresh semen ,106. In The vitrification of fish sperm has recently gained attention as an alternative to the conventional cryopreservation of salmonid sperm ,136,137.The potential benefits of using the cryopreserved sperm of SRF in hatchery practices include the following: (i) Sperm can be preserved and used when the eggs are accessible; (ii) it enables the use of all volumes of accessible sperm; (iii) broodstock maintenance is simplified—out-of-season spawning can be performed only in females and cryopreserved sperm can be used to fertilise the eggs; (iv) it allows the transportation of gametes between distant fish farms and (v) enables the storing genomes of valuable strains of SRF; and (vi) it enables the revitalisation of SRF lines in hatcheries.Gene cryobanking mostly focuses on the storage of cryopreserved sperm of SRFs . Several9 spermatozoa mL−1 is obtained. However, the greatest challenge is the implementation of cryopreserved sperm for mass fertilisation. In hatcheries, the fertilisation of eggs is mainly performed with larger amounts of eggs, e.g., 120,000–140,000. The fertilisation of such amounts of eggs with cryopreserved sperm would require the use of 80 to 93 straws . Recently, sperm samples of gynogenetic rainbow trout, which were cryopreserved in 2016, were used for the revitalisation of rainbow trout lines after two years of storage [The implementation of cryopreserved sperm into the practical conditions of hatcheries is a great challenge. The main problem is scaling up the fertilisation protocols to meet the needs of hatcheries. Recently, it has been demonstrated that it is possible to fertilise up to 1600 rainbow trout eggs using the cryopreserved semen of SRF rainbow trout at a single application without a decrease in the spermatozoa fertilising ability . If we a storage . A totalThe use of high-throughput proteomic techniques for either the profiling or comparative studies of spermatozoa and seminal plasma proteins have successfully been implemented for various fish species (reviewed by ). One ofSalmonid fish spermatozoa acquire the potential for motility during their transition from the testis to the spermatic duct. During this transition, increases in the concentration of bicarbonate and pH take place, which seem to be essential for sperm motility acquisition in rainbow trout . SRF milThe incubation of sperm in ASP resulted in increased sperm motility parameters and had no effect on sperm viability . At the At present, the majority of salmonid fish aquaculture production is primarily based on the use of SRF semen for the production of all-female populations and female triploids. However, despite the significant progress that has been made in the development of sex reversion procedures, the major challenge is still obtaining high quality semen. The major factors influencing semen quality are the season, age of fish, and method of semen extraction from the testes. Moreover, the different lines maintained in hatcheries are characterised by the high variability in semen quality. Therefore, sperm maturation protocols that significantly improve sperm quality are successfully implemented into hatchery practices. Moreover, the development of efficient semen cryopreservation procedures offers a powerful tool for the breeding of SRFs and providing a constant supply of high-quality cryopreserved semen year-round. However, the implementation of cryopreserved semen into hatchery practices is a significant challenge owing to the limited knowledge of fish breeders regarding the use of cryopreserved semen and lack of personnel skilled in performing high-throughput cryopreservation processes and the fertilisation of eggs using cryopreserved sperm.The experience gained during recent years has led to the development of procedures resulting in the production of functional SRFs of salmonid fish. This technique could increase the efficiency of hatchery operations by reducing the accidental harvesting of immature males. Moreover, a protocol based on immersion administration to fish  would simplify the capture of the steroid before it reaches the environment. To the best of our knowledge, the main obstacle for implementing the production of functional SRFs is the necessity to have 100% females for sex reversal . It should be considered that recent progress in the selective breeding of rainbow trout greatly improved the marketable size of fish and may offer an alternative to sex control and triploid induction strategies to produce fish  that can achieve market weight before sexual maturation . This ap


African swine fever remains an important pig disease globally in view of its rapid spread, economic impacts and food implications, with no option of vaccination or treatment. The Southern Highlands zone of Tanzania, an important pig-producing hub in East Africa, is endemic with African swine fever (ASF). From approximately the year 2010, the recurrence of outbreaks has been observed and it has now become a predictable pattern. We conducted exploratory participatory epidemiology and participatory disease surveillance in the Southern Highlands to understand the pig sector and the drivers and facilitators of infections, risk factors and dynamics of ASF in this important pig-producing area. Pigs continue to play a major role in rural livelihoods in the Southern Highlands and pork is a major animal protein source. Outbreaks of diseases, particularly ASF, have continued to militate against the scaling up of pig operations in the Southern Highlands. Intra- and inter-district and trans-border transnational outbreaks of ASF, the most common disease in the Southern Highlands, continue to occur. Trade and marketing systems, management systems, and lack of biosecurity, as well as anthropogenic (human) issues, animals and fomites, were identified as risk factors and facilitators of ASF infection. Changes in human behavior and communication in trade and marketing systems in the value chain, biosecurity and pig management practices are warranted. Relevant training must be implemented alongside the launch of the national ASF control strategy for Tanzania, which already established a roadmap for combating ASF in Tanzania. The high-risk points  and high-risk period (November–March) along the pig value chain must be targeted as critical control points for interventions in order to reduce the burden of infection.  Sus scrofa) and feral pigs are very susceptible to ASF like the domestic pigs but the Warthog (Phacochoerus africanus), the red river hog (Potamochoerus porcus) and bushpig (Potamochoerus larvatus) usually remain asymptomatic [Globally, African swine fever (ASF) has become a disease of great concern, with threats to international trade and food security, especially with the recent expansion of geographical territories to cover previously unreached parts of the world ,2. In 20ptomatic ,4,5,6.The United Republic of Tanzania experienced outbreaks of African swine fever (ASF) since its first occurrence in the country in 1962 . Major eFrom 2016, ASF appeared to have assumed a predictable annual cyclical pattern of infections in different locations in the Southern Highlands, Tanzania, a situation that has forced stakeholders in the pig value chain to adopt modified practices, perceptions, attitudes and behaviors, including the entrenchment of ‘knowledge’ of ASF. Primarily, genotypes IX, X, XV and XVI have circulated widely until 2010, when a virulent genotype II caused outbreak in the Southern Highlands with near 100% mortality ,12. The This work was conducted with the permission of the Director of Veterinary Services, Ministry of Livestock and Fisheries, Dodoma, Tanzania, under the approval number: MA 154/355/16. All respondents were willingly recruited and were made aware of their rights and privileges before enrolment for participation. Each participant was free to discontinue participation at any stage of the process without a notification. All efforts were made to comply with biosecurity protocols and ensured that no farm was contaminated in the process of conducting this study.A desk review of all outbreak reports and recent ASF outbreaks including rumors of outbreaks in the Southern Highlands was undertaken. We received outbreak reports from Veterinary and Livestock Field Officers responsible for the infected and non-infected selected wards. To qualify for inclusion, the village must have been located in one of the districts in the Southern Highlands. The Veterinary and Livestock Field Officers were probed through in-depth questions where details in the reports need clarity and all information obtained were confirmed with the Directorate of Veterinary Services, Ministry of Livestock and Fisheries, Dodoma, Tanzania. Epidemiologic data collected include timelines of outbreaks and rumors, historical outbreaks, formal and anecdotal reports, field interventions, control/eradication efforts, livestock markets and movement patterns. The Veterinary and Livestock Field Officers from selected villages were pre-informed of the planned activities and such officers organized the randomly selected stakeholders for interview (focus group discussions (FGDs) and in-depth interviews (IDIs)). The FGDs, guided by checklist, and the IDIs, developed as categorical questions, were administered to selected stakeholders. Data for the FGDs were collected continuously until the saturation point was reached . The use of categorical data for the IDI was to ease data processing, minimize variation and improve response precision. While the FGDs extended between two and three hours, the IDIs lasted for approximately an hour. Pre-selected veterinarians were trained on the use of participatory epidemiology and participatory disease surveillance (PE/PDS) using available training manuals ,20,21. AIn-depth interviews (IDIs) (n = 37) were conducted with randomly selected farmers, butchers, traders and other suitable stakeholders. These interviews were conducted to triangulate the FGDs, to fill missing gaps arising from the FGDs, and to collect detailed information on the biosecurity and preventative practices at household and community levels. Proportional piling  of the surveyed locations and all other locations mentioned in the discussions were obtained. Information obtained include the following: year of outbreaks, length of outbreaks, regions, districts and wards affected, number of affected animals, number dead, likely source of infections, primary and secondary roads, major livestock (pig) markets, major water bodies , pig population densities, and any other epidemiological details necessary for the outbreak and risk map. All details were submitted to the Geographic Information System (GIS) laboratory, Institute of Resource Assessment (IRA), University of Dar es Salaam, for mapping and outputs were cross verified by field veterinarians.VP72 gene of ASFV using PCR as described by Bastos et al. [During the course of the study, and based on the community (participants’) description of the ASF clinical signs and syndrome , a farm that was experiencing the described clinical signs and syndrome was visited and sampled. A clinically sick but pregnant pig was purchased and euthanised by exsanguination. Spleen, mesenteric and gastro-hepatic lymph nodes, visceral fluid, fetal fluids, uterine fluids, spleen, serum and whole blood from the euthanized pregnant pig; and spleen from a diseased pig during the previous outbreak were collected for ASF confirmation using molecular methods. Samples were aliquoted in the Tanzania Veterinary Laboratory Agency (TVLA) Zonal laboratory in Iringa and dispatched separately to the ASF laboratory in Sokoine University of Agriculture , with ds et al. ,12 and Cs et al. .Descriptive statistics  were determined for all the quantitative values from the community interviews (FGDs), the stakeholders IDIs and details from the proportional piles.We interviewed a total of 95 individual stakeholders in 11 FGDs and 37 IDIs, covering six villages and a public slaughter facility for pigs in two districts of Mbeya and Chunya. Approximately 56.8% of the participants were male and 43.2% were female. Women participated equally with men and moderators of each FGD ensured that crowding-out, drowning of minority opinion and other cause of biases were prevented. Specifically, in 36.4% of the groups, women were the lead respondents and approximately 50% of the veterinary/livestock officers were female. Women dominated 54.5% of the groups by population and one group was almost exclusively women. Where some members of a group appeared to dominate responses, convenient control was brought in by redirection of questions to subdued group members, or discussions were initiated from the apparently weaker group members. Stakeholders were farmers, butchers, slaughter assistants, transporters, traders, and middlemen. The livestock field officers served as opinion shapers but did not influence the exact responses from stakeholders. While five of the communities have experienced previous outbreaks of ASF (2016–2017), one had an ongoing outbreaks (November 2019) and only one of the communities has never experienced outbreaks of ASF based on history and collected anecdotal evidence. Although it was indicated that pigs are kept indoors, during the post-harvesting period of crops, boars are allowed to feed in grains and other crop fields. We also observed a few roaming pigs, especially around the slaughter slabs and near the dump sites. In the Southern Highlands, between 2010 and 2011, outbreaks of ASF were first reported in Kyela district of Mbeya region, which shares international boundaries with Malawi. Legal and illegal trades of livestock occur across the borders and may have facilitated the introduction and transmission. These outbreaks spread inwards and covered seven districts of Mbeya region , and in other districts including but not limited to Temeke, Ilala, and Kinondoni municipalities of Dar es Salaam. Details are available in previous reports [In the Rukwa region, outbreaks have been reported and sustained in 2011, 2014 and 2017. Both home slaughter and the use of slaughter slabs prevailed in Rukwa and Katavi and it is not yet known whether the ongoing outbreaks of 2019 involves the Rukwa region . In IlejA total of 13 animal species featured in the description of commonly found animals at the village level in the Southern Highlands including chickens, cattle, pigs, ducks, goats, cats, dogs, rabbits, sheep, pigeons/doves, guinea fowls, donkeys and guinea pigs. Although there were village by village differences in the ranking of animal populations by proportions in the villages, overall, out of every 100 animals, chickens ranked the highest (29 ± 20) with cattle (21 ± 8), pigs (14 ± 6) and goats (14 ± 5) following in that order. Guinea pigs are the least common animals . In termThe common diseases and syndromes were ranked based on impacts and importance to the economy as follows: (i) African swine fever (69%), (ii) diarrhea ((7%), (iii) mange (10%), (iv) worms (10%), (v) respiratory infections (1%), vi) tremor (0.2%), (vii) abortion (2%), (viii) cyst (1%), and (ix) vomiting (1%) . SimilarThe seasonal calendar indicated that most of the pig-related diseases and syndromes  occurred all the year round , whereasVP72 gene by PCR and RT-PCR, indicating that the farmers’ knowledge of the syndromes and clinical signs associated with ASF is extremely good. Interestingly, samples of fetal tissues and fluids that were aseptically collected during sampling were positive for ASF, indicating transplacental transmission. All the nine samples tested positive by PCR and RT-PCR , recorded for spleen, whole blood and serum.There was a 100% match between signs and syndromes of ASF, as described by the stakeholders and molecular diagnostics based on partial amplification of the ASFV d RT-PCR . All samThe information detailed below arose from the qualitative interviews conducted during the FGDs and the IDIs, backed up by key informants (veterinary and livestock field officers) where detailed information was lacking. The details represent the opinions of the stakeholders and not those of the interviewers or authors.In the Southern Highlands, farmers keep mixed breeds of pigs and villages respond to rumors of fatal disease of pigs promptly. Specifically, pig owners mostly sell off all adult pigs and retain only the piglets, young weaners and pregnant sows. Live pigs were sold to traders (farm-gate buyers) who collect pigs in the farms and slaughter at the village level or in other distant locations, and villagers may also slaughter few pigs. Most of the pig producers refused to scale-up their production system due to fear of annual cycles of ASF infection. Sometimes, farmers with infected herds refused to report to veterinary authorities but simply discarded infected dead carcasses in the community refuse dumps where scavengers (primarily dogs and other pigs) accessed the carcasses. Carcasses buried in shallow pits were sometimes dug up and feasted on by scavengers. To date, the government has no compensation policy for losses associated with ASF infections in farms and, coupled with the fact that there is no treatment for the disease, the farmers’ motivation to report outbreaks is low. While pig viscera  and trotters were sold for local consumption or given to the slaughter assistants for use, other waste material like intestinal contents and scalded materials are dumped in the open refuse site. Due to water scarcity at the rural communities, common water sources  were preferably used in livestock production, both for drinking and cleaning and the community believed that ASF outbreaks were associated with rains which washed infections from upstream areas downstream, or possibly transmitted through the air.A single farmer who operated strict compartmentalization, maintained a close herd in an infected zone, and refused visits from visitors and farm-gate buyers had kept pigs since 2003, with no outbreak to date. Similarly, a community in Chunya, located at an altitude 1700 m, which used both intensive and extensive (scavenging) management systems had incurred economic losses in 2017 associated with outbreaks in other communities but has been free from infection. The significant difference in this community was in the restrictions of all cadres of stakeholders from farm visits during the outbreak period and pigs were slaughtered and shared as pork in the community, but no pork was allowed from outside the community.Overall, biosecurity practices across the surveyed villages were poor. All farms sold their products to farm-gate buyers and raised mixed pig stocks. Approximately 68% and 81% notified neighbors and Veterinary and Livestock Field Officers during ASF outbreaks respectively. A total of 95% preferred communal sale of pig visceral or sharing it with neighbors and only 14% buried gut contents. Only 10% of the farmers implemented any form of quarantine when new pigs were purchased and 19% practiced safe disposal of waste, even though 81% removed manure routinely from the pig pen. Finally, respondents claimed to lock pens (95%), assessed pig health using professionals (92%), not mix age (84%) and not mix species (95%) .(i)Movements of live pigs towards markets sites and around these sites: The identified livestock (pig) markets , Usangu and Uyole) were listed in the consensus map . While s(ii)Movements of live pigs towards slaughtering sites and around these sites: Pig traders and middlemen travel around to buy pigs in bulk from multiple sources, farms and locations . They enter many farms directly to buy pigs and move live pigs from farms to markets and vice versa. Slaughter slabs are quite common, and most villages have privately or public owned pig slaughter facilities. Farmers sometimes bring pigs to the abattoirs/slaughter facilities unsolicited, and if such pigs were not selected for slaughter, they were kept in the human habitations near the slabs as temporary holding grounds, until the next few days when they were returned home. Scavenging pigs may also be seen around the slaughter slab , with im(iii)Movements of pig products from the slaughtering sites (including wastewaters): Purchased pigs can stay up to ten days or more before slaughter depending on the market dynamics. Quite a number of the pig traders also keep pigs at home, and same people handled the home-kept and purchased pigs in terms of feeding and cleaning. During outbreaks and rumors of outbreaks, cheaper prices are offered for sick and recumbent pigs. The farm-gate buyers admitted that they were aware of their contributions to introduction and transmission of infections through transportation of meat using motorcycles and small pick-up vans. They confirmed that they move from slaughter facilities to farms, often with contaminated knives and materials, ready for emergency slaughter, and without any form of disinfection. Public slaughter slabs may have pipe-borne water and supervising veterinarians or veterinary assistants, yet the wastewaters used for washing of carcasses and intestines during slaughter sometimes flowed into the holding tanks and overflowed to contaminate nearby streams . Such st(iv)Movements of people into the slaughtering sites and from the slaughtering sites: People sighted in the vicinity of slaughter slabs include the following: veterinary officers, veterinary assistants, butchers, slaughter assistants, middlemen, traders, farmers, farm-gate buyers, transporters and women who trades in trotters and cook pork. Butchers sometimes sell meat directly to end users (customers) who took them for roasting at a special meat grilling area called “Kitimoto” or homeward for cooking. The intestine, head and trotters are often sold to women who prepared and sold them with the local beer. Such butchers started the day by slaughtering and distributed meat portions to the Kitimoto selling point (mostly pubs) and later went home to clean and feed their own pigs.To provide a summarized overview, the results of the qualitative interviews for this section are as follows:In recent times, traders, transporters and butchers avoid buying pigs in outbreak areas and this action prevented further outbreaks in the 2017 events. The noticed behavioral change was primarily because many of the customers in the area, who also keep pigs, avoided buying pork during outbreaks. Hence, the traders, transporters and butchers were forced to comply with supplying meat from non-outbreak locations to avoid further transmission of infections. The traders were however unaware of the recent outbreaks and suspected cases in Chunya district.a) Humans and anthropogenic issues ;b) Fomites ;c) Animals .The identified risk factors and facilitators can be grouped as follows: 2 [Approximately 46%–60% of all Tanzania’s pigs are found in the Southern Highlands ,24,25. P2 . The app2 . Details2 , and thoThe present work has shed light on the dynamics of African swine fever in the Southern Highlands, Tanzania. For almost a decade (2010–2019), the ASF virus strains inflicted untold economic hardship and decimated livelihoods with severe food security implications. In this work, the understanding of the drivers, risk factors and dynamics of ASF virus circulation in the Southern Highlands from the perspective of the often neglected, yet strategic stakeholders are very important. We interviewed a panel of stakeholders  with a view to gain insights into the pig value chain, socio-economics, behavioral  and anthropological issues that continue to drive and facilitate outbreaks of ASF in the Southern Highlands.Stakeholders in the Southern Highlands considered pigs as very important contributors to rural livelihoods. Although chickens and cattle were present in greater proportions in the Southern Highlands, the economic contributions of pigs cannot be underestimated. Previous workers have confirmed the predominant role of pigs in rural livelihoods in the Southern Highlands ,25,27. IASF was designated as the highest ranked disease by importance and impacts on morbidity and mortality a,b, yet United Republic of Tanzania’s National African swine fever Strategy and Control Plan, 2019’ in February 2020. In the present study, the overall biosecurity systems for the pig value chain in the Southern Highlands were poor. A lot of biosecurity gaps were identified in our analysis and this should warrant the attention of veterinary authorities to safeguard the pig sector of the livestock industry, particularly in the Southern Highlands. Key training on good farming practices, biosecurity principles, risk reduction protocols and safe trade in livestock and its products must be planned and implemented in the Southern Highlands to reduce the burdens of ASF ,18,31. SInterestingly, the stakeholders were experienced enough to know all the syndromic signs and symptoms associated with ASF and all the nine samples collected based on their descriptions were matched 100 using PCR. As such, the surveillance system (both passive and active) can take advantage of stakeholder knowledge to fast track disease diagnosis through prompt reporting. Their modified behaviors on issues of biosecurity, management practices and sales/marketing can be utilized as an entry point to drive home empirical-based interventions . Of partIn addition, although the stakeholders reported that it will appear that less pathogenic strains of ASF virus are in circulation, this will need further evaluation as 1) pathology may be aggravated in naïve infections and may be reduced in subsequent outbreaks; 2) following the index wave of outbreaks, subsequent challenges may be accompanied by modified human behaviors in which the affected farmers may not reports deaths; 3) early interventions in subsequent outbreaks may reduce the impacts observed compared to observations in previous outbreaks. Based on the seasonal calendars, the most common pig diseases occurred all the year round but ASF occurred cyclically from November to March. The months of October to November and January to February were recorded as months with lowest sale of pig and pork; in these periods, the stakeholders are primarily economizing for purposes of cropping in preparation for school fees and other school-related expenses in the following January. December may receive a surge in sale due to festivities (Christmas and New Year) and July to October were reported as months when supply of pigs from farmers are higher, because farmers were trying to earn in preparation for the October and November expenditures on cropping while at the same time minimizing on the risk of losing mature pigs in later parts of the year, the high-risk period for ASF. In the identified but complex calendars , during The slaughter slabs were particularly highlighted as high-risk points for disease introduction and transmission. Other workers have confirmed the major role of the slaughter slabs in the introduction and transmission of ASF ,35,36. IAnecdotally, outbreaks of ASF in neighboring countries of Malawi and sometimes Zambia are a risk factor for infection of border towns in the Southern Highlands in Tanzania. On at least three occasions, outbreaks in Karonga, Malawi, facilitated infections in Kyela and other inland districts of Tanzania . Similarly, outbreaks in Songwe and Tunduma have been linked with previous outbreaks of ASF in the Zambian borders. In view of these observations, the utilization of epi-zonal approach and joint border surveillance for selected high-impact diseases will be a good approach in combating the scourge of ASF in these joint border areas. United Republic of Tanzania’s National African swine fever Strategy and Control Plan, 2019’ already factored in several of the identified risk factors and drivers of infections. Its implementation should therefore benefit the country in its effort at controlling ASF in Tanzania, with benefits for rural livelihoods, food security and household incomes. The national and subnational governments should join forces to combat the endemicity of ASF virus in the Southern Highlands of Tanzania. However, because the government resources are always spread thin in developing economies like Tanzania, international supports from donor partners and efforts from the regional and continental bodies will be necessary to support the government in its effort to combat the unrelenting incidences of ASF in Tanzania. Finally, the stakeholders appreciated the engagement and ensued discussions and requested feedback and follow-up training programs on best practices on livestock husbandry and diseases control/biosecurity.The identified risk factors, drivers and facilitators of ASF infections have been listed in this work. The draft ‘The Southern Highlands of Tanzania remains a strategic hub for pig production in East Africa but the cyclical outbreaks of ASF in this region continue to militate against progress in the sector, especially with the prevailing situation of biosecurity, trade dynamics and management practices. Using the identified issues in this paper, the national , regional and international bodies should join efforts to support the control and eradication of ASF in Tanzania, particularly in the Southern Highlands. Such efforts should involve the policy makers, financiers and resource allocators, stakeholders in the industry, the veterinary authorities and disaster management offices. The issue of ASF should not be looked at from the prism of veterinary services alone, considering its implications on livelihoods, food and nutritional security, rural economies and disruption to regional trades and transboundary potential. We advocate the utilization of a participatory epidemiology and participatory disease search in such control and eradication efforts.


Terpenes are a group of phytocompounds that have been used in medicine for decades owing to their significant role in human health. So far, they have been examined for therapeutic purposes as antibacterial, anti-inflammatory, antitumoral agents, and the clinical potential of this class of compounds has been increasing continuously as a source of pharmacologically interesting agents also in relation to topical administration. Major difficulties in achieving sustained delivery of terpenes to the skin are connected with their low solubility and stability, as well as poor cell penetration. In order to overcome these disadvantages, new delivery technologies based on nanostructures are proposed to improve bioavailability and allow controlled release. This review highlights the potential properties of terpenes loaded in several types of lipid-based nanocarriers  used to overcome free terpenes’ form limitations and potentiate their therapeutic properties for topical administration. Thus, e.g., hemiterpenes (hemiterpenoids) are formed by one isoprene unit, monoterpenes (monoterpenoids) have two isoprene units (C10), sesquiterpenes (sesquiterpenoids) have three (C15), and diterpenes (diterpenoids) have four (C20) isoprene units. Terpenes may also be classified as linear, monocyclic, and bicyclic [Terpenes and their oxygenated derivatives terpenoids are the largest and most common class of secondary metabolites. Terpenoids are modified terpenes where methyl groups are moved or removed, or additional functional groups  are added. The two terms are often used interchangeably. They are found in higher plants, as representing the majority of molecules in essential oils, mosses, liverworts, and algae lichens, as well as insects, microbes, and marine organisms ,2,3. Thebicyclic . Smallerbicyclic .Targeted delivery of typically hydrophobic terpenoids has been the subject of much research, finding applications in a wide variety of fields. As naturally occurring compounds, they have been applied in transdermal research since the 1960s and are reported to be a safe and effective class of penetration enhancers ,9. PlentLipid-based nanocarriers are novel drug delivery systems that have been widely explored for topical and transdermal delivery of pharmaceuticals. Encapsulation of terpenoids in such a system is an interesting strategy to provide better stability and protection against environmental factors that may cause chemical degradation. In addition, nanoencapsulation can decrease the toxicity, solubilize poorly soluble terpenes, improve bioavailability, and achieve controlled and sustained delivery, in addition to drug targeting at the site of action ,16.In the present review, the topical administration of selected terpenoids is discussed with special emphasis on nanostructured delivery systems applied as carriers for these groups of bioactive compounds.The topical administration of bioactive compounds acting as drugs relies on the localized administration of formulations to a body through dermal and mucosal  routes. Skin is one of the most easily accessible organs in the human body for topical administration and is the major route of localized drug delivery ,17. The The intact skin is much less permeable than other tissues and penetration of the active compounds depends on the physicochemical properties of the penetrant, the condition of the skin, and the nature of the carrier . TopicalDrug administration through mucosal and transmucosal routes like oral, nasal, and ocular is still a challenge for scientists mainly because of the presence of mucus, saliva, and lacrimal fluids, which can restrict the access of bacteria or virus to the deeper layers but can also affect the bioavailability of bioactive compounds like terpenes ,19.Topical terpenoids application can be an attractive route of administration as it allows the reduction of side effects, avoids the injectable route, the first-pass metabolism, and pre-systemic elimination within the gastrointestinal tract. Moreover, it is a safe route with the easiness of application and fast action .The effectiveness of transdermal drug delivery depends on the sufficient capability of drugs to penetrate through the skin to reach the therapeutic level. An important barrier of the skin for drug absorption is the stratum corneum (SC) ,22. The Terpenes and terpenoids have attracted great interest as effective enhancers from natural products ,24,25,26Terpenes acting as permeation enhancers are usually used as excipients in formulations  and are The most widely investigated therapeutic potential of terpenes and terpenoids for topical administration is the anti-inflammatory activity, including burns or wounds healing ,12,13,14Inflammation is the physiological response of the body to tissue injury , microbial and viral infections, or genetic changes and may have an acute or chronic state . The mosAn equally important therapeutic aspect of terpenes is their activity against skin cancer. The number of skin cancer cases has increased rapidly worldwide. Skin cancer, including melanoma and non-melanoma skin cancer (NMSC), represents the most common type of malignancy in the white population ,54. The The choice of vehicle or delivery system in the case of skin diseases has a significant influence on the outcome of topical dermatological drug treatment. Nanoparticulate systems can improve the stability of actives in front of possible degradation by light, heat, and other environmental factors. Moreover, they provide better bioavailability, improve permeation through the skin and other biological barriers, and reach the controlled delivery of drugs ,66. ThesNanoemulsions (NE) are thermodynamically stable oil in water (o/w) or water in oil (w/o) dispersions stabilized by surfactant molecules. The mean droplet size, ranging from 20 to 200 nm, and the low percentage of surfactant make them ideal for topical drug delivery with reduced skin irritation . NEs areLiposomes are vesicles built by amphiphilic lipids, mainly cholesterol and phospholipids. The lipids arrange themselves in bilayers surrounding an aqueous core, where hydrophilic compounds can be entrapped while hydrophobic drugs can be encapsulated on the bilayer. Liposomes are very versatile drug delivery systems since they can incorporate either hydrophilic, hydrophobic, or amphiphilic drugs ,69. LipoLipid nanoparticles are innovative nanocarriers known as solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) and represent a revolution in the efficient encapsulation of hydrophobic drugs and long-term physicochemical stability of lipid-based drug delivery systems . They arVesicular systems have been among the most studied for topical therapies during recent years . The phyNiosomes are a form of liposomes composed of nonionic surfactants that produce more stable, less toxic, and more flexible vesicles. They are also less expensive and more economical to manufacture . NiosomeEthosomes are elastic vesicles composed of phospholipids, cholesterol, water, and large amounts of ethanol that help to improve the solubility of lipophilic drugs and aid in disrupting the SC. Therefore, the delivery of drugs into the deep dermal layers or even into the systemic circulation is possible ,82. WhenInvasomes represent vesicular carriers for enhanced skin delivery. They are composed of unsaturated phospholipids, small amounts of ethanol, terpenes, and water. Different penetration studies performed in vitro in human skin were represented in order to show the penetration-enhancing ability of invasomes. They are characterized by elasticity and deformability, which favors penetration across skin layers ,28,29,84Nowadays, phytochemicals like terpenes and terpenoids have promising potential to prevent and treat different types of diseases. Although several terpenes are considered GRAS, few studies investigated the safety of these compounds in direct topical application. Their poor water solubility, stability, and bioavailability, as well as other side effects , have limited their clinical application. New drug delivery systems based on lipids represent an encouraging approach for topical applications of terpenes. These systems are attractive, non-invasive, and especially beneficial for patients that are not viable for surgery or highly intensive non-specific systemic therapies. Encapsulation of terpenes in lipid-based nanocarriers is widely described in the literature as an approach providing protection against environmental factors that can cause chemical degradation and volatilization of these compounds. Moreover, nanostructured lipid systems allow controlled drug release and enable the passage of the bioactive compounds through biological barriers making them ideal candidates for topical applications. Finally, terpenes and lipid-based nanosystems represent a sustainable alternative in pharmaceuticals, giving increasing importance to greener chemistry.


The extent of ribosomal heterogeneity has caught increasing interest over the past few years, as recent studies have highlighted the presence of structural variations of the ribosome. More precisely, the heterogeneity of the ribosome covers multiple scales, including the dynamical aspects of ribosomal motion at the single particle level, specialization at the cellular and subcellular scale, or evolutionary differences across species. Upon solving the ribosome atomic structure at medium to high resolution, cryogenic electron microscopy (cryo-EM) has enabled investigating all these forms of heterogeneity. In this review, we present some recent advances in quantifying ribosome heterogeneity, with a focus on the conformational and evolutionary variations of the ribosome and their functional implications. These efforts highlight the need for new computational methods and comparative tools, to comprehensively model the continuous conformational transition pathways of the ribosome, as well as its evolution. While developing these methods presents some important challenges, it also provides an opportunity to extend our interpretation and usage of cryo-EM data, which would more generally benefit the study of molecular dynamics and evolution of proteins and other complexes. The ribosome is a large and universal RNA-protein complex that mediates protein synthesis. In recent decades, progress in imaging technologies fueled considerable advances in understanding its atomic structure. While first structures at a near-atomic resolution were established using X-Ray crystallography ,2,3, theThe purpose of this review is to present the recent advances made in the quantification and extent of ribosome heterogeneity. In the first part, we will cover the multiple aspects and scales of heterogeneity, from the dynamical aspects of ribosomal motion, to differences of composition across species. As these various aspects of heterogeneity call for new methods and tools, we will focus in the second part on the computational challenges currently faced to exploit the information available from cryo-EM data. Overall, the development of these new computational methods for analyzing structural heterogeneity promises exciting new insights for the study of the ribosome and its biological implications.Since the first structures described in 2000 that led to the Nobel Prize in Chemistry in 2009, the ribosome has been a central focus of structural biology, with more than 500 structures published since 2015 see . During As recent ribosome structures account for all domains of life and diverse families, they provide an important way to study heterogeneity across species. To illustrate this diversity, we report 20 different species for which ribosome cryo-EM structures have been recently published in Recent studies of the ribosome composition and structure for diverse species have contributed to draw a more intricate picture of the ribosome evolution. An important example of divergence among eukaryotes is the kinetoplastid family, which has been the object of several structural studies ,22,23,24As sequence variability carries major differences in the ribosome structure across species, single mutations offer another source of structural heterogeneity within them. Without the need for crystallized structures, the structural and functional consequences of these modifications can be elucidated by cryo-EM. Although testing for every possible nucleotide mutation is a daunting task, focusing on key functional regions allows one to reasonably mitigate it. For example, a first standardized and complete mutational survey was recently produced for the Peptidyl Transferase Center (PTC) , a regioS. aureus erythromycin resistant mutant [Acinetobacter baumannii [While this approach has been relatively recent in human ,42,43,44t mutant , or fromaumannii , a Gram-aumannii ,53,54.The scope of ribosome heterogeneity also expands within cells and across tissues and cell types. Paralog or alternative ribosomal protein and rRNA genes provide a direct source of ribosome heterogeneity . The extIn situ visualization of repaired or defective structures by cryo-EM would help to confirm this hypothesis but also reveals challenging, as one needs to generate enough samples for 3D reconstruction and classify the different particles according to these modifications. Cryo Electron Tomography (cryo-ET) provides an exciting direction for visualizing the ribosome in situ, e.g., interacting with organelle membranes or as parts of polysomes ,66,67. TTranslation involves major conformational changes of the ribosome, which gets assembled and translocates to the next codon at each elongation cycle. To capture these changes, as well as those of many other cotranslational processes, cryo-EM offers the ability to separate millions of sampled particles into multiple volume classes, which provide snapshots of the ribosome dynamics. From these different conformational states, one can infer a wide range of motions, such as multiple rotations relative to the LSU or the SSU , displacDespite offering an increasingly detailed view of the ribosome at different stages of translation processes, these multiple conformational states of the ribosome structure still offer a static overview of the conformational landscape. Yet, detailed 3D structures can serve as an important basis or complement for more direct studies of the underlying kinetics. Coarse-grained and atomistic molecular dynamics simulations take 3D structures as an input to model the thermodynamic and kinetic properties of the ribosome ,87. On tUnraveling all of the aforementioned aspects of ribosome heterogeneity poses various computational challenges. This in turn has made the ribosome the center of modern developments in cryo-EM.ex.PF01248, PF00467) span multiple nomenclature classes, there remains a need for manual curation and disambiguation. By the same token, certain proteins belong to multiple PFAM families based on their sequence and some remain unclassified. These many-to-many mappings along with the differences in the classification methods employed by member-databases of InterPro make a fully-automated conversion mechanism between PFAM/InterPro and the proposed nomenclature (While the plethora of available structures makes a comparative analysis of ribosomal structures timely, performing such studies proves challenging in practice. First of all, in order to compare structures deposited by the community in a shared data bank , a commonclature e problemIn light of these issues, there is a need for ribosome-centric databases that gather available 3D structures, associated protein data at multiple structural scales, and allow users to compare ribosome components across these structures. In addition, an important needed feature would be to provide enough flexibility to augment such databases with the publication of new structures. Previous efforts were made to build databases and interfaces for 3D alignment structures , or joinA detailed comparative analysis of the ribosome structures can help to elucidate the extent and implications of the diversity of the ribosome and the various degrees of homology of ribosomal RNA and proteins ,97. SimpFrom an evolutionary perspective, the diversity of cryo-EM structures also allows one to treat the ribosome geometry at the molecular level as a quantitative trait, and thus establish direct association between conservation of structures and sequences. For such a complex and heterogeneous 3D object as the ribosome, it is yet challenging to find metrics that can properly detect evolutionary variations as done for sequence-based phylogenies. Our recent study of the geometry of the ribosome exit tunnel can be seen as a first attempt to do so . AlthougAs highlighted in the first part of this review, the development of cryo-EM has led to determine multiple structures of the ribosome that reflect its conformational heterogeneity. The most popular computational method used to address conformational heterogeneity is referred to as 3D classification in most standard softwares used for single particle reconstruction ,8,108 anBeyond discrete classification, the construction and inference of continuous motion is an important goal for improving our understanding of molecular behavior from cryo-EM data. Morphing-based techniques can be used to interpolate and visualize continuous trajectories between classes. In particular, our lab recently developed a morphing tool suited to perform transport-based interpolation between EM maps , while pWith unknown pose and microscope parameters, high signal-to-noise ratio, and limited sampling of particle images, the context of cryo-EM image formation makes this problem challenging. Yet, multiple approaches have been proposed to approximate the manifold of heterogeneous conformations. A few groups have proposed ways to approximate the manifold with a linear subspace, akin to principal component analysis ,114, and