Bovine tuberculosis (bTB) was first diagnosed in cattle in South Africa in 1880 and proclaimed a controlled disease in 1911. Testing of cattle for bTB is voluntary and only outbreaks of disease are reported to the National Department of Agriculture so the prevalence of the disease in cattle is largely unknown. There is a Bovine Tuberculosis Scheme which is aimed at the control of bTB in cattle but the same measures of test and slaughter, and the quarantining of the property apply to wildlife as well. bTB was first diagnosed in wildlife in a greater kudu in the Eastern Cape in 1928 and has to date been found in 24 mammalian wildlife species. The African buffalo has become a maintenance host of the disease, which is considered endemic in the Kruger National Park, the Hluhluwe-iMfolozi Park and the Madikwe Game Park. Control of bTB at the wildlife-livestock interface is difficult because of spill-over and spill-back between species. Only buffalo are required by law to be tested before translocation, but bTB has been introduced to the Madikwe Game Park probably by the translocation of other infected wildlife species. There is no national control strategy for the control of bTB in wildlife. Indirect tests have been developed to test for bTB in eight species, 6 of which can be considered endangered. More research needs to be done to develop an effective and efficient vaccine to combat the transmission of bTB within and between species. New policies need to be developed that are effective, affordable and encompassing to control the spread of bTB in South Africa. South Africa is a land of beauty and splendour, it is also a land of diversity and contrast. South Africa ranks as the third most biodiverse country in the world with 95,000 known species, 299 of which are mammalian . There aAccording to the World Health Organisation (WH0) an estimated 360,000 cases of active human tuberculosis (TB) occurred in South Africa in 2019. The incidence has been dropping since 2009 but TB still remains the leading cause of death in South Africa , 6. In aClimate change is adversely affecting South Africa – dry seasons are becoming longer, and the rainy seasons are starting later and becoming more variable. Torrential downpours as opposed to soft soaking rains cause flash flooding , 8. MuchCattle farming practices are many and varied in South Africa ranging from commercial farmers, many employing first world standards; to smaller commercial units, emerging farmers, communal  farmers and subsistence farmers. Commercial cattle make up around 58% of the national herd, emerging, communal and subsistence cattle 42% of the national herd of 13,601 million cattle. Dairy cattle account for approximately 10% of cattle in the commercial cattle population , 12. TheThe Animal Diseases Act, 1984 (Act 35 of 1984) makes allowances for the control and eradication of bTB through its regulations and “Bovine Tuberculosis Scheme R1953”. Testing is voluntary and reporting is poor. Little attention is paid to livestock other than cattle. There is no political will, or funding to support the scheme. There are no subsidies granted for TB testing. The scheme does not differentiate between the control of bTB in cattle or wildlife, or between commercial and communal farmers. There is a socio-economic difference in the value of cattle belonging to commercial and communal farmers which is not considered in the current Bovine Tuberculosis Scheme , 15.The purpose of this rather bleak introduction is to bring into perspective the challenges that can face veterinarians in their quest to either perform a TB test, their interpretation of the TB test once performed; and the control and eradication of the disease.Positive skin reactions to the SICTT, or macroscopic lesions indicative of M. bovis infection in culled animals are reported to the epidemiology section of Veterinary Services in Pretoria. The division in turn verifies the data and from this data, Table During January – December 2019, 28 outbreaks of bTB comprising 64 cases were reported to national government.Case - means an individual animal infected by a pathogenic agent, with or without clinical signs. Outbreak - means the occurrence of one or more cases in an epidemiological unit. Where an epidemiological unit means a group of animals with a defined epidemiological relationship that share approximately the same likelihood of exposure to a pathogenic agent .Whilst during the Covid-19 pandemic a very different picture emerged.The prevalence of bTB in both cattle and wildlife is unknown.There are in essence 3 Acts that provide the legal mandate for control actions related to bTB namely the Animal Diseases Act, The Meat Safety Act and the Animal Identification Act.Mycobacterium bovis, M. avium and M. tuberculosis. As a result of the Act, a voluntary “Bovine Tuberculosis Scheme, R1953” was promulgated on 30 September 1988 and is presently still applicable. There was a uniform computerised reporting system across the four provinces as well as reliable and standardised census data. The bTB Scheme is a voluntary scheme, but once bTB is diagnosed or suspected in a herd, control becomes legally compulsory and a quarantine on the movement of cattle is imposed. There is a Veterinary Procedural Notice for Buffalo disease risk management  for the testing of buffalo for bTB prior to movement. There is no legislative requirement to test other wildlife for bTB.Bovine tuberculosis was first noted in cattle in South Africa in 1880 and has been a notifiable animal disease in South Africa since 1911. The “Bovine TB eradication scheme” was introduced in the Republic on 14 May 1969. At that time there was political will as well as sufficient funding of the scheme, and sufficient manpower to implement the scheme. There was a high level of compliance on the part of commercial farmers for the scheme. During 1970/71, 232 Stock inspectors were trained, and private veterinarians were contracted by the state to test for bTB. The state paid compensation at slaughter for any cattle diagnosed on the intradermal skin test as being infected with bTB. When the Animal Diseases Act, 1984 (Act 35 of 1984) replaced the previous Animal Diseases and Parasite Act, 1956 (Act 13 of 1956), tuberculosis was maintained as a controlled disease. The Animal Disease Regulations (GN R2026 of 1986) that was promulgated in terms of Act 35 of 1984, refers to the control of After 1994 when South Africa became a democratic republic with nine provincial governments, disease control was devolved to the provinces. The control of bTB became a lesser priority with funding prioritised for other priorities within provinces. In accordance of the Animal Diseases Act, control is based on test and slaughter in domestic species and buffalo. Although provision for compensation is made in the Animal Diseases Act, compensation is generally not paid.ante-mortem inspection, primary and secondary meat inspection. Primary meat inspection means the inspection, by a registered meat inspector, of a carcass and organs directly after flaying and evisceration. Secondary meat inspection means the inspection, by a registered veterinarian, of a carcass and organs detained during primary meat inspection such as cattle with visible tuberculous lesions and especially anergic cattle with advanced lesions. The ideal is that when identified during primary meat inspection performed at abattoirs there will be traceability back to the farm of origin.According to the Meat Safety Act, 2000 (Act 40 of 2000), no person may slaughter any animal at any place other than an abattoir except if the meat is for their own consumption or for cultural purposes. It is acknowledged that Iillegal or informal slaughtering does take place of which infected TB carcases will not be notified to local authorities. The owner of the abattoir must procure a registered independent meat inspector to perform a meat inspection service. The meat inspection includes In terms of the Animal Identification Act, 2002 (Act 6 of 2002), there is a herd identification system where cattle should be branded or tattooed with a unique identification mark supplied by the Registrar of the Act. This Act is policed through the Stock Theft Act, 1959 (Act 57 of 1959) by members of the various Provincial Stock Theft Units, a specialised unit of the South African Police Services. In theory this unique brand mark should be able to trace an animal found to be infected with tuberculosis back to a herd of origin, but in practice it rarely does as animals may not be marked, they may have changed ownership or marks may be unidentifiable. Very few cases of bTB are diagnosed at an abattoir, and if diagnosed are rarely traced back to the herd of origin. There is legislation available to support a national animal identification and traceability system (LITS) in the country under the Animal Identification Act, 2002, but this has not been implemented yet. This will hopefully address one of the challenges of tracing cattle found to be infected with bTB at an abattoir back to the farm of origin.In 1994 South Africa had its first democratic elections. Nine provinces were created, and veterinary services provincialized with central government having a concurrent responsibility with the provinces for animal disease control but in reality, only overseeing and setting of national policies for disease control. Each province has its own focus for animal health and disease control. The OIE summarised the problems arising from provincialisation in their Performance of Veterinary Services (PVS) Evaluation report of the Veterinary Services in South Africa dated October 2012. They reported “Constitutional change has introduced a break in the chain of command in the VS (Veterinary Services) as it has become the “concurrent” responsibility of both national and provincial political authorities. This break in command is universal except in cases of national emergency”. This break in command has also negatively affected disease reporting. The OIE report goes on further to state that “Data management is generally effective and widely utilised. However, the break in the chain of command limits data collection, analysis and reporting at central level. The data is not being used to develop comparative, efficacy, efficiency and cost benefit analyses for animal health programmes” [Currently the National Department of Agriculture does not know how many non-infected herds are being tested for TB as only infected herds are reported by provinces. Because of this and poor census data, the prevalence of bovine TB cannot be established and therefore strategies to implement surveillance or to control and eradicate bovine TB cannot be developed and implemented. Currently there is no national strategic plan for the control and eradication of bTB in livestock or wildlife, and generally no compensation for the slaughter of infected animals.The breaks in the chain of command negatively affects authority and the manner in which the Bovine Tuberculosis Scheme is implemented in each province. Currently the compliance of livestock owners with the applicable legislation for bTB testing, and the enforcement thereof by government is variable. All bTB testing apart from infected herds and some surveillance cases is paid for by the cattle owner. There is otherwise no funding by industry or the state. Dairy farmers are required by legislation to test their herds for TB, this mainly as a result of export requirements, so the majority of dairy cattle are tested. Some milk buyers will deduct a small amount per litre of milk from payment if the TB and Brucellosis tests are not up to date. This acts as an incentive for the dairy farmer to test. It is thought that most of the estimated 1,4 million dairy cattle in the country are tested for bTB . Stud bePresently there is no clear national implementation plan, or political will to eradicate bTB from cattle herds or wildlife in South Africa. In recent years attention has been focussed on the control of African Swine Fever (ASF), Highly Pathogenic Avian Influenza (HPAI) and Foot and Mouth Disease (FMD) that are draining resources and place exports in jeopardy. As previously stated, the Bovine Tuberculosis Scheme is a voluntary scheme, and implementation of the current legislation is inconsistent and insufficient. National government is focussing on education, social protection and human health, not Agriculture, resulting in a low prioritisation of funding for veterinary service delivery. The Department of Agriculture, Land Reform and Rural Development (DALLRD) only received R27.5 billion of the total budget of R2,157.3 billion in the 2022/2023 budget allocation or 1.2% of the 2022/23 National budget .Furthermore, there are many vacancies and unfunded posts in State veterinary services. Newly qualified veterinarians are obliged to conduct compulsory community service for the first year after graduating as veterinarians in the employ of national government, either in national government or provincial posts. This is an attempt to fill these vacant positions, not contributing significantly to TB testing. Private veterinarians and Animal Health Technicians (AHTs) working in the private sector are testing cattle and herds, but they are not contracted by the state to do so which leads to poor reporting and low testing frequency. Authorisation of veterinarians and AHTs is needed to stimulate compliance with reporting.M. bovis is part of the Mycobacterium tuberculosis complex (MTC) that constitutes a remarkably genetically homogeneous group. bTB probably has the widest host range of the pathogenic mycobacteria. MTB and bTB are characterized by a 99.9% similarity at the nucleotide level and identical 16 S rRNA sequences [equences . As statequences –25.M. bovis and M. tuberculosis. Confirmatory testing on all cultured mycobacteria using an immunochromatographic test (ICT) based on the detection of IgG antibodies is used. If the ICT is negative then molecular tests are performed to identify the species of the organism, which includes M. bovis, M.caprae and BCGosis. These cases are extremely rare and given the burden of M. tuberculosis in South Africa this differentiation is not considered a priority . Muller et al. [M. bovis is resistant to one of the antibiotics used [Zoonotic tuberculosis can occur due to closer contact with cattle with a higher potential infection rate in untested animals and the absence of pasteurisation of milk and its products, wound mediated transmission during slaughter, or the consumption of infected undercooked meat. The higher level of poverty, malnutrition and HIV-infection rate decreases immunity in humans making them more susceptible to infection . If cattr et al.  made no ics used .M. tuberculosis was cultured as a co-infection with M. bovis from tonsillar tissue in a buffalo from the Northern Cape Province [M. tuberculosis has been cultured from a Chacma baboon living in the wild  and in a wild elephant in the Kruger National Park .A reverse zoonosis is also possible as management practices bring cattle into close contact with MTB, and it will sensitise cattle to the bovine Purified Protein Derivative (PPD) tuberculin and interfere with skin test results . This seProvince . M. tubeM. bovis, may not be responsive on the skin test (anergic) and remain in the herd spreading disease [Infected cattle with advanced pathology due to bTB and which are more likely to be shedding  disease –31. The  disease . The ELISouth Africa does not manufacture its own bovine or avian tuberculin PPD, so this needs to be imported through an import permit acquired from the registrar of the Fertilizers, Farm Feeds, Seeds and Remedies Act, 1947 (Act 36 of 1947). Often this permit can take months to acquire leading to a shortage of tuberculin in the country, and a halt to testing. Currently bovine tuberculin PPD of two manufacturers can be imported into the country – from Intertest (through MSD), and Prionics . Intertest’s concentration is 50,000 IU/ml , and PriBoth PPD tuberculin should be stored at a temperature of 2–8˚ Celsius, but Prionics PPD may be transported at 2–37˚ Celsius for up to 14 days which may lead to misinterpretation by the end user . The stoMycobacterium to survive. Water is a scarce commodity in many areas of South Africa and is being recycled and used to clean the concrete aprons in front of the parlour. Other methods of cow management may also negatively impact on disease control. The damp moist conditions found in dairies can allow for the growth of bTB and other Non-Tuberculous Mycobacteria (NTM) which can sensitise the animal and interfere with the skin tests [In dairy herds the risk of spread of bTB between cattle increases as not only do dairy cattle congregate at the dairy parlour two to three times a day, but they also congregate around feed bunkers and water troughs. This risk can be influenced by management where some dairies are moving to a system of zero grazing, and herd housing with fans and misters to keep cows cool, creating the optimum environment for the in tests , 36. FroIn urban and rural areas, cattle are often kept in small camps close to dwellings at night due to the risk of theft or predation, this congregation of animals increases the risk of spread of bTB in an infected herd and contact with MTB and NTM . The catMycobacterium malmesburyense was cultured from various places in South Africa from water, nasal and pharyngeal swabs, as well as bovine lymph nodes [A novel NTM, ph nodes . Skin reph nodes , 36.As the skin test has to be assessed 72 h after the introduction of PPD, sometimes in the case in emerging, communal or subsistence production systems some or all the animals are not presented for palpation and diagnosis. To try and avoid this situation, incentives are often used e.g. treatment with anti-parasitics to rid the cattle of either internal or external parasites during reading.In large dairies, individual cow numbers are normally not recorded – numbers will only be recorded when done in conjunction with blood sampling for bovine brucellosis. Numbers of cattle tested are therefore given by the farmer from his computer spread sheets and may not be accurate. There are often duplicate numbers when other herds with the same format for numbering cows are bought in. Quite often cattle may inadvertently not be tested, or not be presented after 72 h as they have changed milking groups or calved down. As milk production drops when dairy cattle are taken out of their routine, farmers get annoyed with the loss of income over the testing period.Routine or parallel Interferon gamma (IFG) blood tests to determine the presence of bTB in a herd are not routinely done or are not practical because of cattle numbers, distance to laboratories and great expense involved. Recent research conducted using a bacteriophage method combined with phage-PCR demonstrated MTC circulating in blood . This meCattle belonging to farmers in urban and deep rural areas are not used to being handled which makes the process of testing and reading tedious. Pens and crushes are often inadequate or absent. Mobile pens and crushes have been purchased in some state veterinary areas to overcome these difficulties. Long distances, setting up and handling times have a negative effect on the efficiency of TB testing and the use of non-thermal stable PPDs. These cattle are often not individually identified. Another challenge encountered is that torrential rains can cause rivers and streams to become impassable or wash away infrastructure so reading the test 72 h later, or doing follow up testing becomes difficult. The harsh summer sunlight causes sunburn on unpigmented skin which can interfere with the interpretation of skin tests.In some communal areas one must arrange for testing through the tribal chief, headman or community leader. Negotiation is often very complex and time consuming. If any infected animals are found, retesting of the communities’ cattle is challenging as socio-economic dynamics cause resistance to further testing. Cattle may also have been moved to better pastures for grazing during times of drought . CulturaTragelaphus strepsiceros) and common duiker (Syvicapra grimmia) in the Eastern Cape. The first case diagnosed in the Kruger National Park (KNP) was in an impala (Aepyceros melampus) in 1967. In 1970 bTB was diagnosed in a black rhinoceros (Diceros dicornis) in the Hluhluwe-iMfolozi Game Park (HiP) in Kwa Zulu Natal. During 1990 bTB was diagnosed in the African buffalo (Syncerus caffer) in the south of the KNP and has since spread to the north of the park and into neighbouring Zimbabwe. bTB has also been diagnosed on private game farms and ranches. The presence of well-established national and provincial game parks as well as the increasing development of wildlife ranches in South Africa, is increasing the wildlife-livestock interface and risk of either wildlife contracting disease from livestock (spill-over) or livestock contracting disease from wildlife (spill-back) [M.bovis has been diagnosed in 24 of the 246 terrestrial mammalian wildlife species in South Africa [Bovine TB was reported in South African wildlife as early as 1928 when it was diagnosed in a greater kudu (ll-back) , 17, 38.h Africa .Wildlife host species can be described as maintenance hosts where infection can persist without introduction from another source, or spill-over hosts when species become infected incidentally , 17. As M. bovis, Brucella abortus, Foot and Mouth disease (FMD) as well as Corridor disease caused by the protozoan Theileria parva lawrencei. All four diseases are transmittable from buffalo to cattle, and freedom from these diseases is to prevent spread of disease, and protect other buffalo farms as well as cattle at the wildlife-livestock interface. Buffalo needs to be chemically immobilised for testing and confined in a boma. Confinement in bomas when testing may bring buffalo into contact with NTM which may influence the outcomes of the SICTT [African buffalo movement is strictly controlled by the Buffalo VPN  with Single Intradermal Comparative Tuberculin Test (SICTT) testing required before movement between farms or game parks with negative results. Each farm or park owner that wants to keep buffalo must apply for registration with national government prior to receiving buffalo, in terms of Regulation 20 A (2) of the Animal Diseases Act, 1984 (Act No. 35 of 1984) as published in Government Notice No. R. 2358 of 10 December 1993, and if approved the farms/parks will receive an individual BU  registration number. All buffalo must be microchipped before testing to ensure individual identification and traceability. Ear tags may be used in conjunction with microchips for easier identification. Pre movement testing of buffalo requires testing for he SICTT , 37. If he SICTT . Often ahe SICTT . Since MThe African buffalo is the most widely known maintenance host of bTB in South Africa . They arThe greater kudu has a social structure, but herds are very small ranging from 3 to 15 individuals with males often being solitary individuals, joining the herd during the mating season. Kudu are browsers so they are primarily infected via the oral route with initial pathology in the lymph nodes of the head where abscessation occurs. Despite its smaller herd size, the greater kudu has the recently been characterised as a maintenance host , 15  bushbuck (Tragelaphus scriptus), nyala (Tragelaphus angasii), blue wildebeest (Chonnochaetes taurinus), eland (Taurotragus oryx), and giraffe (Giraffa camelopardelis).bTB has been confirmed in other South African ruminants including impala, springbok (Phcochoerus africanus) and bushpigs (Potamochoerus porcus) are omnivorous and have been found to be infected with bTB. Warthogs live in burrows and are social animals. They become infected via the aerosol droplet route or per os when scavenging [Warthogs , leopards (Panthera pardus), cheetah (Acinonyx jubatus) and wild dog (Lycaon pictus) have been found to be infected with bTB [per os), and the third is percutaneous when fighting over an infected kill. Lions have the potential to become a maintenance host [Predators such as lion , white rhinoceros (Ceratotherium simum), large spotted genet (Genetta tigrina), banded mongoose (Mungos mungo), Chacma baboon (Papio ursinus), honey badgers (Mellivora capensis) and hippopotamus (Hippopotamus amphibius), and are all considered spill-over hosts [Other wildlife that has been found infected with bTB are elephant (er hosts . Again, M. bovis in eight species of wildlife have been developed [Indirect diagnostic tests to detect cell mediated immunity to eveloped . These teveloped .Vaccination can be a method for controlling the spread of bTB by increasing an animal’s immunity to disease. Vaccination does not necessarily have to prevent infection but should reduce transmission in the host species and reduce spread to other species. There are many challenges to the successful development of an effective vaccine. A trial done on yearling buffalo in the KNP using a BCG vaccine found that there was no significant reduction in the number of lesions or severity of disease . An effibTB is of great significance in wildlife as it is a threat to South Africa’s rich biodiversity with the possibility of localised extinction of some species where mortality is high, and population is low. This will have a serious impact on ecosystems as each species fulfils a function in an ecosystem , 15. TheEradication by test and cull in low prevalence herds/groups of animals would slow down the spread of the disease and may not elicit such an emotional outcry as blanket culling. However, this remains a challenge in large buffalo herds in the KNP . Both teIn conclusion, bTB is a problem both in cattle and wildlife in South Africa with the potential to reinfect both cattle and wildlife at the wildlife-livestock interface. The extent of bTB in humans is unknown as no differentiation between MTB and bTB occurs routinely. bTB can be treated successfully in humans although some drug resistant bTB forms may occur . The pre


However, how dopamine depletion affects the oscillation dynamics of BG nuclei is still unclear. With a spiking neurons model, we here capture the features of BG nuclei interactions leading to oscillations in dopamine-depleted condition. We highlight that both the loop between subthalamic nucleus (STN) and Globus Pallidus pars externa (GPe) and the loop between striatal fast spiking and medium spiny neurons and GPe display resonances in the β range, and synchronize to a common β frequency through interaction. Crucially, the synchronization depends on dopamine depletion: the two loops are largely independent for high levels of dopamine, but progressively synchronize as dopamine is depleted due to the increased strength of the striatal loop. The model is validated against recent experimental reports on the role of cortical inputs, STN and GPe activity in the generation of β oscillations. Our results highlight the role of the interplay between the GPe-STN and the GPe-striatum loop in generating sustained β oscillations in PD subjects, and explain how this interplay depends on the level of dopamine. This paves the way to the design of therapies specifically addressing the onset of pathological β oscillations.Motor symptoms of Parkinson’s Disease (PD) are associated with dopamine deficits and pathological oscillation of basal ganglia (BG) neurons in the  β ([12–30] Hz) frequency range. What is unclear is the mechanism through which dopamine depletion leads to these oscillations. In this work we developed a BG network model and we found that the actual reason for these abnormal oscillations is the synchronization of two loops within the network that are individually oscillating in the β range. For high levels of dopamine the two loops are decoupled and the oscillation power is low. Conversely. when dopamine is depleted (as in PD) the two loops synchronize and originate the pathological oscillations associated with motor symptoms.PD is associated with the death of neurons generating a particular neurotransmitter: the dopamine. Motor symptoms of PD, on the other hand, are known to be due to dysfunctions in a particular subcortical area of the brain: the BG network. In particular, the BG network develops pathological oscillations in the  The basic architecture of the BG network see  consistsβ oscillations [12 − 30] Hz emerge in the STR  Hz , D1, D1νext 020] Hz , GPe-TI020] Hz , GPe-TA020] Hz  and STN020] Hz .Note that the GPi and the SNr nuclei have not been included in the simulated model since they do not present direct feedback to the other populations in the BG and hence cannot contribute to the generation of oscillations.aeif_cond_exp model in the code implementation)  interval  opto-inhibition of motor cortex input, ii) both opto-inhibition and opto-excitation of STN activity, iii) opto-inhibition of GPe activity. Coherently, we analyzed the behavior of our model in pathological conditions (Dd = 1.03) in response to the following activity perturbations.To reproduce the conditions described in  we needei) as a reduction in the mean of the poissonian external input to the STN and a portion of the striatal populations. Since the dendritic domain of individual subthalamic neurons covers large parts of the whole nucleus  so that the activity of STN varies in the range  Hz, coherently with the conditions considered in   the intensity of β oscillations goes to zero in the large n limit;in case of low synchronization (ε ∼ 1) the intensity of β activity is preserved.in case of high synchronization   higher values are registered since the activity of the STN nucleus becomes more affected by the STR oscillator  decreases again as the two oscillators synchronize to a novel frequency which is different from both the natural ones , the increase of Dd determines the convergence of the mean frequencies of oscillation of the STN and D2 populations , the intensity of β activity is preserved , the intensity of β activity decreases , the intensity of β activity decreases .In accordance with previous results, this proves that the intensity of ε = 0.00) and coupled (ε = 0.75) cases highlights that the increase in the intensity of β activity is stronger in the latter case , the system gains temporary access to the synchronous condition and the two oscillators continuously switch between the synchronous and asynchronous states. Moreover, when the value of Δf(t) = fSTN(t) − fD2(t) decreases (meaning that the system spends more time in the synchronous status), the intensity of the β oscillations increases , indicating the presence of transient bursting. Therefore β bursts, although uncommon, are possible, even for low levels of dopamine depletion. On the other hand, when the synchronization occurs, the intensity of β activity increases and the distribution does not show any tail (skewness between -0.29 and 0.29). In this condition, high β power intensity is stable and not transient anymore. These results are in accordance with the previous considerations and indicate once again that the more the two oscillators interact, the more the system spends time in the synchronous state and the higher is the intensity of β activity within the network.One of the most important feature of pathological rization , 21. As β oscillations originate from the synchronization of a striatopallidal loop and a subthalamic-pallidal loop mediated by the GPe. The relevance of GPe in the generation of β oscillations was recently proved by a work of Mallet group  Hz and a high β range [20–30] Hz  Hz –104. Eve agonist  and DBS  agonist  seem to S1 TextTable A. Parameter changes from the Complete to the Simplified Model. Fig A. Effects of the coupling strength ε on the Simplified Model: plots for the D2 nucleus. Fig B. Effects of Dopamine Depletion Dd on the Simplified Model (ε = 0.75): plots for the STN nucleus. Fig C. Effects of Dopamine Depletion Dd on the Complete Model: plot for the STN nucleus. Fig D. Effects of the connectivity alterations due to Dopamine Depletion on the intensity of β activity. Fig E. Robustness of the results to alterations of the connectivity parameters and neuron and population parameters. Fig F. Probability distribution of the instantaneous intensity of STN activity for different degrees of Dopamine Depletion.This file collects the following supporting tables and figures: (PDF)Click here for additional data file.


Additionally, this work provides comprehensive vibrational frequency calculations and assignments for Finasteride, Lamivudine, and Repaglinide, which have never been thoroughly investigated in previous research.By means of a computational method based on Density Functional Theory (DFT), using commercially available software, a novel method for simulating equilibrium geometry harmonic vibrational frequencies is proposed. Finasteride, Lamivudine, and Repaglinide were selected as model molecules to study the adaptability of the new method. Three molecular models, namely the single-molecular, central-molecular, and multi-molecular fragment models, were constructed and calculated by Generalized Gradient Approximations (GGAs) with the PBE functional via the Material Studio 8.0 program. Theoretical vibrational frequencies were assigned and compared to the corresponding experimental data. The results indicated that the traditional single-molecular calculation and scaled spectra with scale factor exhibited the worst similarity for all three pharmaceutical molecules among the three models. Furthermore, the central-molecular model with a configuration closer to the empirical structure resulted in a reduction of mean absolute error (MAE) and root mean squared error (RMSE) in all three pharmaceutics, including the hydrogen-bonded functional groups. However, the improvement in computational accuracy for different drug molecules using the central-molecular model for vibrational frequency calculation was unstable. Whereas, the new multi-molecular fragment interception method showed the best agreement with experimental results, exhibiting MAE and RMSE values of 8.21 cm Infrare−1 ,4. With −1 .The vibrational frequencies serve as a distinctive signature for chemical compounds, and are commonly employed in the identification and characterization of organic and inorganic specimens ,8,9. TheIn recent years, the application of DFT in the field of pharmacy has experienced a rapid progression ,14,15. QHowever, up to the present, all of the research works have been optimized and frequency-calculated by constructing single-molecular structures with different functionals and basis sets ,24. The The development of scale factor calculation has been divided into two stages. In the first stage, the factors were determined as an average of the experimental/theoretical ratios for individual modes utilizing numerous molecules. For instance, Hout, Levi, and Hehre (1982)  determinω-B97X-D, can better describe anharmonic effects, the accuracy of the results is limited by various drawbacks of the single-molecular model. While the GGA/PBE method we use does not directly consider anharmonic effects in predicting vibrational frequencies, it is still a very common computational method in this field at present [Harmonic approximation assumes that vibrations are harmonic and linear, which simplifies calculations but has severe limitations when it comes to describing real-world vibrational phenomena . The har present ,21,22. TThere are several reasons why including multiple molecules in our calculations can help reduce the limitations of the harmonic approximation and improve accuracy. Firstly, a multi-molecular model can take into account intermolecular interactions, which are crucial in many chemical processes, such as hydrogen bonding or protein–ligand binding. Neglecting such interactions can lead to significant errors in spectroscopic predictions. Secondly, in actual multi-molecular systems, the interactions between different molecules can change the frequency and intensity of vibration modes. For example, in a multi-molecular hydrogen-bond model, hydrogen bonding typically leads to a significant enhancement of anharmonic effects in the vibration modes and introduces higher-order vibration terms . When weIn this paper, a novel vibrational simulation method was developed to facilitate the correct assignment of infrared spectra. The pharmaceutical molecules Finasteride (FIN), Lamivudine (LAM), and Repaglinide (REP) were employed as models for this purpose. The theoretical and experimental results of the vibrational harmonic spectra were compared to validate the new method. Three models were constructed in the Material Studio program, and vibrational simulations of single molecules, multi-molecular fragments, and central molecules were conducted and compared with the scaled spectrum.Frequency simulations conducted thus far have only involved single molecules and scaling with scale factors. However, calculated spectra with scale factors still exhibit significant deviation from experimental results, and the scale effect varies across different molecules and functional groups, even with the same factor. To verify this problem, we extracted FIN, LAM, and REP single molecules as model molecules, optimized and calculated their harmonic vibration frequencies and infrared spectra, and compared these with the experimental spectra. The infrared spectra for the experimental spectra, the simulated single molecule, and the simulated single molecule after scaling for FIN, LAM, and REP are displayed in −1 and 29.80 cm−1, 52.17 cm−1 and 109.50 cm−1, 26.48 cm−1 and 57.51 cm−1 for the single-molecular simulation; and 16.99 cm−1 and 27.14 cm−1, 50.56 cm−1 and 101.75 cm−1, 29.39 cm−1 and 44.34 cm−1 for the scaled spectra of FIN, LAM, and REP, respectively. Therefore, after scaling, the infrared spectrum of LAM exhibited greater similarity to the experimental data, while the opposite was observed for FIN and REP. Thus, for macro-molecules, particularly those with complex configurations, the scale factor may not be entirely applicable. Nonetheless, at hydrogen-bonding sites, the scaled vibrations outperformed the single-molecular simulations. In the case of FIN  by the central-molecular model. Therefore, the central-molecular model for vibrational simulation was found to be more accurate than the single-molecular and scaled results for all three pharmaceutics. Moreover, for the hydrogen-bonded functional groups, the central-molecular model was also found to yield better fits to the experimental spectra.From After optimization, the geometrical parameters  of the single molecule may differ from those of the central molecule due to the lack of intermolecular interactions in the former case. For instance, in the case of the REP molecule, the ethoxy and carboxyl groups were twisted by 4.06 and 6.22 degrees, respectively, after optimization by the single-molecular and multi-molecular repeating units  among different models, we classified the vibrational frequencies of three types of drug molecules into functional group vibrations and non-functional group vibrations. We then studied in detail the changes in AEs caused by different models, as shown in The results showed that for functional group vibrations, the scale factor had some accuracy improvement effect on the correction of the single-molecular model results, but the effect was generally poor and fluctuated greatly. In the non-functional group set, the scale factor correction increased the calculation error, instead of lowering it. Therefore, the mechanism of adding scale factors sacrificed the calculation accuracy of skeletal vibrations to promote a higher fit between functional group vibrations and experimental values, although this increase in accuracy was not significant see in .In addition, for functional group vibrations, although the center-molecular model comes from the optimized structure of multi-molecular models, it does not contain intermolecular hydrogen-bonding, or conjugated, information, but the center molecule model has a significantly reduced error compared to the single molecule model. This may be due to two reasons. First, the center molecule model inherits the advantages of the multi-molecule model, and the averaged molecular structure composed of multiple molecules is more similar to the actual molecular structure. Therefore, by averaging multiple molecules and taking the center molecule, the compression or deformation of the actual molecular structure caused by strong interaction forces such as hydrogen bonding can be eliminated. Secondly, considering the intermolecular interactions together with the individual atomic structure in the optimization process can improve the accuracy of calculating the frequency of hydrogen-bonding functional groups in the center molecule model. However, the center molecules of REP did not show small relative AE values, and the center molecule models of FIN and LAM molecules showed great differences in accuracy improvement. This indicates that there is still considerable instability in using center molecule models for vibrational frequency calculation. For the non-functional group region, the analysis of the AEs between the center molecule and the multi-molecule fragment model shows that the AEs of the two models are very similar, and the decrease in AEs compared to the single molecule model is also similar. This indicates that the improvement in calculation accuracy of the non-functional group region is mainly due to the fact that the molecular structure is closer to the true state after optimizing the overall molecular structure of the multi-molecule repeating unit, and the effect of hydrogen bonding information on the vibrational calculation optimization of the non-functional group region is not significant. This conclusion is also easy to understand because there is no hydrogen-bond generation in the non-functional group region, so the addition of multi-molecule hydrogen bonds has a very small effect on vibrational calculation.−1, 8.96 cm−1 and 57.04 cm−1, 18.94 cm−1 and 28.36 cm−1, 13.36 cm−1 in the functional group and non-functional group regions, respectively. For LAM and REP, the improvement of computational accuracy in the functional group regions had a more significant effect on reducing the overall MAE, whereas for FIN, the contribution of both functional group and non-functional group vibrations to the overall error was similar. Interestingly, for all three drug molecules, the magnitude of MAE reduction in the multi-molecular fragment model in the functional group region (LAM > REP > FIN) was consistent with the variational trend of intermolecular hydrogen bonds or interactions. This indicates that, for multi-molecular models, the more intermolecular hydrogen bonds present, the greater the reduction in errors observed in functional group vibrations. This may be due to the retention of hydrogen bonding and conjugation information in the multi-molecular model, leading to higher computational accuracy at the hydrogen-bonding connection sites during vibrational frequency calculation.Additionally, we can also illustrate that for FIN, LAM, and REP, the reduction in MAE values of the multi-molecular fragment model and single-molecular scale model were 8.07 cmIn conclusion, the improvement in accuracy of the center-molecular and multi-molecular fragment models in the non-functional group region was mainly due to the molecular structure being closer to the true state, and the molecular conjugation information having no significant effect on the accuracy improvement of this part. In the functional group region, both molecular structure and intermolecular conjugation information had significant impacts on the calculation accuracy of vibrational frequencies. The more hydrogen bonding among molecules in the original structure, the greater the contribution of intermolecular conjugational systems to the reduction of MAE. However, the center-molecular model did not have good accuracy performance in all drug molecule models, while the multi-molecular fragment model showed the lowest error values among all models for all drug molecules.To provide a quantitative comparison of the computational performance among different models, we conducted structural optimization and vibrational frequency calculations for all models using the same computer and computational resources. The time required for each calculation is summarized in Traditional single-molecular models have the smallest number of atoms, and requires the least amount of time for configuration optimization and vibrational frequency calculation, with a total calculation time not exceeding 3 h. In our paper, the central-molecular and multi-molecular fragment models both used minimal molecular repeat units with hydrogen bonds as initial structures for configuration optimization, causing a significant increase in the time required for this step. Compared to the single-molecular model, FIN, LAM, and REP required an additional 3.91 h, 15.30 h, and 27.95 h, respectively. In the vibrational frequency calculation stage, the central-molecular model took a similar amount of time as the single-molecular model because only one molecular unit was involved. Whereas, due to the inclusion of a large number of hydrogen bonds and conjugated structural fragments in the multi-molecular fragment model, its frequency calculation took approximately 2 h longer than that of the single-molecular model.However, when selecting drug molecules as calculation models to demonstrate the applicability of new methods more convincingly, we specially selected drug molecules with a high number of atoms, complex structures, and representative hydrogen-bond numbers. For small drug molecules, commonly used for vibrational analysis verification, such as aspirin, the calculation time using the multi-molecular fragment model was only 0.29 h. Moreover, we were pleased to find that although the overall calculation time was longer due to various reasons, including the lack of professional computing servers, our calculations could still be completed within roughly one day. Achieving high computational accuracy is critical in ensuring precise spectral analysis, as even small infrared spectral peak shifts can cause significant analytical errors. Thus, considering the significant reduction in MAEs and the instability observed in the prediction results of the central-molecular model , we regaThe key findings from our study also have several important implications for molecular simulation and drug design. Firstly, the improved accuracy of the multi-molecular fragment interception model suggests that accurately describing intermolecular interactions is critical for accurate vibrational frequency calculations. This finding highlights the importance of advanced modeling approaches that accurately capture the complex interplay between molecules in biological systems. Secondly, this method can be applied to a wide range of molecular systems and provide insights into their structural and functional properties. By accurately calculating vibrational frequencies, the new approach can aid in the interpretation of IR spectra and elucidation of molecular structure and function. This capability is particularly relevant in drug design, where understanding the vibrational properties of drug molecules is critical for predicting their behaviors in vivo. Additionally, this method provides highly accurate guidance for the determination of supramolecular structures, such as co-crystals or co-amorphous systems, enabling precise structural characterization. Thirdly, it has potential applications in optimizing drug formulations and identifying potential drug candidates with desired properties. By accurately describing the intermolecular interactions that contribute to the stability and efficacy of drug formulations, the new approach can aid in the development of more effective and efficient drug delivery systems. Additionally, by accurately describing the vibrational modes associated with specific functional groups, this method can facilitate the identification of potential drug candidates with specific properties or functional groups. We believe that this approach has significant implications for molecular simulation and drug design, and has the potential to contribute to the development of more effective and efficient drug design and development strategies.Finasteride, Repaglinide, and Lamivudine were obtained from Hunan Qianjin Xiangjiang Pharm. Inc. . Ethanol was obtained from Sinopharm Chemical Reagent Co., Ltd. . The chemicals were used as received from the companies without further purifications.The Finasteride form I  and RepaThe Lamivudine form II  was prodThe precipitates were stored in a desiccator until use in the experiment.−1. Samples were prepared in KBr pellets by grinding Ca. 1 mg of the drug with KBr and the resolution was 2 cm−1.FT–IR patterns were recorded using a NICOLET 380 FT-IR spectrometer  at wavelengths of 4000–400 cmThe spatial configuration and atom numbering schemes of Finasteride, Repaglinide and Lamivudine molecules are given in For each pharmaceutical molecule, we created three distinct models: the single-molecular model, the central-molecular model, and the multi-molecular fragment model, with the single-molecular model serving as a control group. The single-molecular model was a single molecular structure obtained by optimizing the molecular structure extracted from the crystal cell. The central-molecular and multi-molecular fragment models were optimized with multi-molecular hydrogen-bonded repeating units. The minimum multi-molecular hydrogen-bonding repeating unit refers to the random selection of a central molecule and the extraction of all the molecules that were hydrogen-bonded to it to form a multi-molecular hydrogen-bonding unit, with the aim of preserving the molecular spatial structure and hydrogen-bonding information as much as possible. We obtained the central molecule by taking the central molecule after optimizing the multi-molecular repeating unit with the GGA/PBE functional , a widel−5 Hartree, 0.002 Hartree Å−1 and 0.005 Å, respectively. The vibrational frequencies of the single-molecular and multi-molecular fragment were simulated at the same level of theory as the geometrical optimizations, and the MAEs between the calculated and experimented frequencies were compared to validate the accuracy of the new method. All calculations were conducted by the DMol3 module in Material Studio 2017 program package [The structural optimization of the single-molecular and minimum repeating units was performed at the GGA/PBE functional level under vacuum conditions. PBE is the recommended default exchange–correlation functional, especially for studies of molecules interacting with metal surfaces, but also widely-used and reliable for bulk calculations . Converg package , utiliziIn summary, our study presents a novel methodology for simulating equilibrium geometry harmonic vibrational frequencies using multi-molecular fragment interception models. The results showed that the traditional single-molecular simulation and scaling method deviated significantly from the experimental results, while the central-molecular model was better but still not accurate enough, especially for hydrogen-bonded functional groups. Moreover, using the central-molecular model for vibrational frequency calculation resulted in unstable computational accuracy for different drug molecules. The multi-molecular fragment model, on the other hand, demonstrated the highest calculation accuracy with minimum simulation deviation, by preserving the configuration information and intermolecular interactions. The study also provided complete vibrational frequencies calculations and assignments for the three pharmaceutics, which could provide more accurate interpretations of infrared spectra and have never been thoroughly investigated in previous research. Overall, the proposed method has the potential to subvert the current frequency simulation method and improve the accuracy of vibrational spectrum interpretation.Despite the potential applications of the method, there are limitations and challenges that must be overcome to further enhance its accuracy and applicability. One limitation is the computational cost of the multi-molecular fragment interception model. The model requires a large number of calculations to account for intermolecular interactions, which can increase computational time and limit its applicability to larger systems. To overcome this limitation, one possible improvement would be to incorporate machine learning approaches to optimize the multi-molecular fragment models and improve computational efficiency. For example, artificial neural networks could be trained to estimate intermolecular potentials, reducing the number of calculations required to accurately describe intermolecular interactions. Another limitation is the scalability of the methodology to larger molecular systems. The multi-molecular fragment interception model is more accurate than traditional single-molecular simulations and scaling methods but has limitations in accurately capturing intermolecular interactions in large systems. To overcome this limitation, one possible future direction would be to explore the use of hybrid functionals, which combine the strengths of different exchange–correlation functionals, to improve the accuracy of vibrational frequency calculations for complex molecular systems. By incorporating these hybrid functionals, we may be able to accurately describe intermolecular interactions in larger systems, expanding the applicability of the method to a wider range of molecular systems.


Schizophrenia (SZ) is an inherited mental illness that affects 1% of the world’s population. Schizophrenia is a common functional psychosis with some unifying features that appears to have a universal distribution. Much of the research on SZ seeks to identify physiological, biochemical, or genetic features that differ between patients and healthy individuals. Biochemical factors represent an imbalance of certain biochemical substances in the brain, especially neurotransmitters. Early studies focused on the brain biochemistry of patients with SZ in terms of dysregulation of the neurotransmitter network. There is evidence that elevated dopamine concentrations are associated with positive symptoms  of the disease. For example, L-DOPA decarboxylase (DDC) is an enzyme involved directly in dopamine and serotonin synthesis and indirectly in noradrenaline synthesis. Therefore, the DDC gene can be considered a candidate gene for schizophrenia and its activity is a good candidate for functional analysis via epigenetic repression.We aimed to create a cellular model with a deregulated DDC gene.We constructed two lentiviral vectors, one expressing the dCas9-KRAB-MeCP2 repressor under the control of a synthetic tetracycline inducible promoter, and the other carrying a cassette expressing two sgRNAs with spacers against the DDC promoter. The SH-SY5Y cell line was sequentially stably transduced with both lentiviral constructs, and cells carrying both constructs were selected by the fluorescence of the GFP and RFP reporter proteins encoded in the lentiviral construct backbones.Methyl-sensitive real-time PCR followed by high-resolution fusion showed methylation of the DDC promoter. Correspondingly, real-time PCR showed a two-fold decrease in DDC expression in SH-SY5Y during tetracycline-induced expression of the CRISPR repressor.We developed a cellular model to study the contribution of DDC deregulation to SZ-related molecular mechanisms.None Declared


The burden of antimicrobial resistance (AMR) is projected to be highest in Africa, with a mortality of 99 per 100 000 deaths in sub-Saharan Africa. As a result, there is an urgent need to develop education resources to raise awareness and improve understanding of AMR.The aim of this review was to evaluate selected games, and inform regarding their suitability, and that of gamification in general in promoting education, and inspiring action against AMR.This brief review of online education resources seeks to inform on the use of games in promoting education on AMR by exploring different aspects of gamification such as accessibility, usability and playability with the delivery of desired learning outcomes. The insights obtained from the game helped inform recommendations and conclusions on how to best utilize gamification to deliver AMR education to target audiences.The games to be reviewed were selected using search terms ‘AMR Game’, ‘Antimicrobial Resistance Game’, ‘Antibiotic Resistance Game’, ‘ABR Game’, ‘Drug Resistance Game’ and ‘Superbugs Game’ in Google Play Store and Apple App Store search engines, given their positioning as suitable application software that house game applications. After applying a selection criterion, the number of selected games was narrowed to two: Micro-Combat and Terebra.The games were analysed through four key factors: accessibility, usability, playability and learning outcomes. Both games were found to apply visual, written and aural game mechanics but Terebra had an increased touch with reality, enhancing its playability, due to the emphasis on aural triggers positioning AMR as a dire health threat. On the other hand, Micro-Combat was found to be more educative, and learning-oriented but less associated with reality, which negatively influenced its playability.In overall, gamification was found to be a good online resource to promote education on AMR through the review conducted on the two games, Terebra and Micro-Combat. Coupling gamification and conventional education mechanisms can go a long way in promoting the awareness and knowledge level of AMR among diverse populations The two games, Terebra and Micro-Combat, are great pilot AMR gamification projects that have set a good pace for utilization of games in AMR education. There is a need to develop AMR educational games that portray the reality in low- and middle-income countries, which was a bit lacking in the two games. One of the objectives of the Global Action Plan on AMR adopted by the World Health Assembly is to improve awareness and understanding of AMR. This is through effective communication, education and training. Recently, there has been an increase in leveraging learning through the use of games, which involves conversion of learning concepts to suitable game designs contextualized to the target audience. It is commonly referred to as gamification.Antimicrobial resistance (AMR) is currently among the leading causes of death and morbidity in the world, with an estimated 1.27 million deaths in 2019 attributed to bacterial infections. In this study, the burden of AMR was projected to be highest in sub-Saharan Africa, with a mortality of 99 per 100 000 deaths.2 A study conducted in Saudi Arabia reported that gamification had the potential to improve AMR knowledge with better retention than the conventional lectures.3 Games are a good educational avenue for different populations and can be harnessed not only to create awareness but also inspire action in the fight against AMR. However, for them to deliver on these two mandates, they have to be suitably designed. The aim of this review is to evaluate selected games, and gamification in general, to inform on its suitability in promoting education and inspiring action against AMR.Game-based learning has been found to improve performance and motivation in learning.The objectives of this educational resource review are: (i) to investigate the different aspects of gamification such as accessibility, usability, playability, and learning outcomes in the games selected, and their role in promoting learning; and (ii) to recommend, and draw possible conclusions about, gamification as an avenue for creating awareness and inspiring action in the fight against AMR.The games to be reviewed were selected using search terms ‘AMR Game’, ‘Antimicrobial Resistance Game’, ‘Antibiotic Resistance Game’, ‘ABR Game’, ‘Drug Resistance Game’ and ‘Superbugs Game’ in Google Play Store and Apple App Store search engines, given their positioning as commonly used software that house game applications. A total of six games were identified in the first stage .In the second stage, the screening of the six games was conducted, and four of the games were eliminated from consideration since they were not directly connected to AMR and provided very limited learning outcomes on the subject. The third and final stage involved an analysis of the games selected. This has been illustrated as a flow diagram in Figure 4 These three key factors, alongside the accessibility of the game, were applied in reviewing the games selected. The two games selected were Terebra and Micro-Combat. Terebra is an individual puzzle game where the player assumes the role of a scientist racing against severe drug-resistant epidemics by creating new antibiotics. The faster the player develops a new antibiotic the larger the population of the world saved. The game becomes more challenging as you go up the levels as the bacteria become more resistant. On the other hand, Micro-Combat is a card cooperative game where the players assume the role of healthcare personnel preventing infection by pathogenic agents through the use of preventive measures and medication.The effectiveness of learning from a game can be seen as a collective measure of accessibility, usability, playability and learning outcomes.Both games are freely accessible via Google Play Store or Apple App Store, and their development was funded. Micro-Combat has a higher number of downloads (1000+ category) compared with Terebra’s 100+ category. This is attributable to the intentional marketing strategy employed in Micro-Combat. No information was found regarding a marketing strategy for Terebra. Terebra is compatible with a lower version of the operating system and was released earlier in 2017 compared with Micro-Combat’s release in 2020. Micro-Combat is available in 19 languages while Terebra is only available in English, making it inaccessible for non-English-speaking audiences. Terebra is classified as a puzzle game rated for 12+ years while Micro-Combat is classified as a card game rated 3+ in Play Store but rated as 10+ years on the game’s website. Both games are therefore suitable for diverse populations.2 Micro-Combat has a personalized account system enabling cooperation between different players. This can help track attendance, harness problem-solving skills, and improve ownership of AMR challenges.5 On the other hand, Terebra is a puzzle game with simple instructions. Puzzles have been found to foster problem-solving, analytical and memory skills. Game-based learning has also been found to be influenced by instructional strategies.6 Here, we looked at the location and nature of instructions. Micro-Combat’s instructions are inbuilt into the game itself while Terebra’s instructions are sequestered in the Google Play game description rather than inside the game, making it difficult for a player to locate them, which could be disappointing and negatively affect engagement in the game. Both games have audio-visual and written instructions. From experience in playing the game, the authors found that it was easy to miss out on Terebra’s instructions, which could affect the player’s motivation. Micro-Combat’s instructions were also difficult to comprehend compared with Terebra’s. Difficult-to-understand instructions can undermine a player’s understanding of the game. Overall, Terebra had better usability compared with Micro-Combat. The usability of a game helps the player understand how to navigate the game, and therefore is an important aspect to review when evaluating utilization of games as education resources. If a player finds it difficult to understand how to play a game, it is likely that they will be disappointed, and spend less time engaging in the game, which negatively affects the intended learning outcomes.The usability of a game involves the level of ease with which the player understands and plays, promoting their immersion in the game.7 There are different frameworks used to evaluate the playability of the game but the background lies in the challenge, mastery and reward mechanisms of the game.8 This guides the design of the game and influences the level of immersion the player experiences.The playability of a game involves a game’s educational capability while still retaining its immersive capacity and motivation.Micro-Combat's game challenge lies in the use of limited preventive measures and medications to prevent characters from getting infections, while Terebra’s challenge is developing a new peptide antibiotic in a drug-resistant epidemic setting amidst a fast-declining world population. Both games address the current threat of AMR but from different perspectives; Micro-Combat addresses the need to improve disease prevention measures in reducing spread of infections, while Terebra focuses on development of novel antimicrobials to avert the drug resistance crisis. Both games apply visual, written and aural game mechanics but Terebra has an increased touch with reality due to the emphasis on aural triggers such as sirens, helicopter evacuations, wails and emergency news briefs, which insinuate a serious pandemic, and trigger the need for urgent action. However, it is less educative on ways to act to avert AMR. On the other hand, Micro-Combat is educative with a broad perspective on processes leading to the development and spread of drug-resistant infections and ways to act, such as potential mitigation strategies that can be employed. The limited time for intervention in each game promotes a higher demand for auditory, visual and mental attention, promoting immersion in the game.9 Extrinsic motivation stems from behaviour that is pegged on the desire to attain an outcome that is separate from the action itself. On the other hand, intrinsic motivation refers to engaging in satisfying behaviour that is independent of external incentives. Both intrinsic and extrinsic motivation have a huge influence on the gaming experience. In both Terebra and Micro-Combat, intrinsic motivation arises from the feeling of responsibility to alleviate the challenges expressed in the game setup. In both games, one feels the nudge to act to prevent the looming AMR crisis projected in the game’s design. In Terebra one feels intrinsically motivated to develop new antibiotics, while in Micro-Combat there is a nudge for continued playing to ensure better infection prevention and therapeutic interventions to avert AMR.9 Micro-Combat exhibits extrinsic motivation through score rewards and trophies, but Terebra has no incentives.Games can have both extrinsic and intrinsic motivation, which is important in maintaining optimal concentration, and engagement, which is crucial in promoting long-duration playing of the game. Ultimately, more time spent in playing the game is likely to improve the learning outcomes from the game.Learning outcomes of a game evaluate the game’s capability to promote education, in this case education on AMR. Micro-Combat promotes learning of scientific concepts on development, the spread of drug-resistant infection, and alleviation measures that can be employed. However, there is less focus on issues such as systemic challenges, and behavioural tendencies that promote irrational use of antimicrobials. The less specific focus in Micro-Combat may undermine positioning of major enablers of AMR as intended game’s learning outcome, especially for audiences from LMICs.10 These AMR drivers thrive due to poor health systems and the poor purchasing power of many LMICs. The AMR drivers are not addressed in the game, and this could undermine players’ inspiration to initiate action on mitigating AMR after playing the game, especially if there are from LMICs.Terebra stirs emotions and positions AMR as a dire health threat. This is likely to have both positive and negative outcomes. It can facilitate the retention of information for a longer period but it could also undermine the credibility of information shared, and discourage the need to take action due to its apocalyptic nature. Terebra does not address the drivers of AMR, and the development of new antibiotics is postulated as the magic bullet to solving AMR, which is not the case in reality. Poor access to already existing antimicrobials, irrational use of the antimicrobials, and poor infection prevention and control strategies are among major drivers of AMR, especially in LMICs.The authors spent an average of 15 min playing one game of Terebra before losing and restarting again. They had an average of four replays for two sessions of playing the game. At the beginning, the duration of play was short before the players understood the game well but the duration increased with time as they got to understand the game better. On the other hand, it took them an average of 25 min to play Micro-Combat, with an average of four replays in two sessions. Despite their different game designs, both games were generally challenging but exciting.Overall, gamification was found to be a good online resource to promote education on AMR through the evaluation conducted on the two games, Terebra and Micro-Combat. Generally, the two games are great pilot AMR gamification projects, which have set a good pace for utilization of games in AMR education. However, for both games, it is important to consider a good balance in the diversity of the information shared to ensure there is an envisioned targeted learning outcome from the games. Educative games should also have the capability of inspiring action or positive change among the players. Intentional efforts should be made to ensure reach of the games to a wider target population, and adaptation to diverse contexts. An evaluation strategy is also important to inform on the attainment of the learning outcomes and recommend potential future improvements in utilization of gamification in AMR education. We recommend further in-depth reviews of these two games reviewed in this paper, and other existing AMR games, to determine the potential and significance of gamification and game-based learning in AMR education and inspiring action. The rapid mobile connectivity and internet penetration in sub-Saharan Africa, and other LMICs, positions gamification as a strategic tool in promoting education of AMR in these settings. There is a need to develop educational games that portray the reality of AMR in LMICs. Both Micro-Combat and Terebra have been developed in high-income countries, which makes the relationship with the situation in LMICs quite distant. Lastly, we recommend initiation of studies that evaluate coupling of gamification and conventional education mechanisms in promoting the awareness and knowledge level of AMR among different populations.


The role of NKG7 expression in the occurrence of treatment-free remissions (TFR) should be evaluated in future studies.The classical natural history of chronic myeloid leukemia (CML) has been drastically modified by the introduction of tyrosine kinase inhibitor (TKI) therapies. TKI discontinuation is currently possible in patients in deep molecular responses, using strict recommendations of molecular follow-up due to risk of molecular relapse, especially during the first 6 months. We report here the case of a patient who voluntarily interrupted her TKI therapy. She remained in deep molecular remission (MR4) for 18 months followed by detection of a molecular relapse at +20 months. Despite this relapse, she declined therapy until the occurrence of the hematological relapse (+ 4 years and 10 months). Retrospective sequential transcriptome experiments and a single-cell transcriptome RNA-seq analysis were performed. They revealed a molecular network focusing on several genes involved in both activation and inhibition of NK-T cell activity. Interestingly, the single-cell transcriptome analysis showed the presence of cells expressing NKG7, a gene involved in granule exocytosis and highly involved in anti-tumor immunity. Single cells expressing as granzyme H, cathepsin-W, and granulysin were also identified. The study of this case suggests that CML was controlled for a long period of time, potentially  Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm initiated by the occurrence of the Philadelphia chromosome Ph1) in a primitive hematopoietic stem cell. The natural history of disease from a chronic phase towards accelerated and blast phases has now been drastically modified by the introduction of the targeted therapies. BCR::ABL1 tyrosine kinase inhibitors (TKIs) improved overall survival in patients with chronic phase CML  in a pri.Up to 80% of newly diagnosed CML patients in chronic phase (CP) achieve a molecular response under TKI therapies especially by the use of second-generation TKI such as Nilotinib . A majorIS levels. In addition, a single-cell transcriptome analysis of the circulating mononuclear cells was performed.We report here the case of a patient who voluntarily stopped her TKI therapy and remained in deep molecular response without therapy for 18 months. At the end of this period of 18 months, which was operationally a TFR, a molecular relapse was diagnosed, but the patient declined therapy for an additional 3 years until hematological relapse. A transcriptome analysis was performed at several time points of this TKI-discontinuation condition with low or high BCR::ABLIS levels, which rose to 3%. She disclosed at that time that she was not taking Dasatinib. Despite this molecular recurrence, she declined therapy while accepting regular hematological and molecular analyses. BCR::ABLIS levels rose to 25% in October 2017 and to 38% in September 2019 , which induced a major molecular response (MMR) that is currently ongoing.A CML was diagnosed in 2014 in a 30-year-old female patient with low Sokal score. She was initially treated with Imatinib, but this drug was rapidly stopped because of intolerance, and she was switched to Dasatinib Figure 1IS quantification was performed, peripheral blood mononuclear cells have been processed to perform total RNA extraction and whole transcriptome experiment on microarray chip ClariomS human (Thermo Fisher Scientific).To gain insights into the molecular events occurring during this “self-induced” drug interruption, we have performed with the informed consent of the patient, serial transcriptome analyses in frozen peripheral blood cells (PBMCs). The first three were performed during periods of low BCR::ABL expression, whereas the last four were performed in samples with high BCR::ABL expression Figure 1IS quantifications were available. BCR::ABL-IS quantification was taken as predictor for Pavlidis Template Matching algorithm (-IS (-IS quantification and transcriptome time-course experiments.Although this patient declined therapy for a long period of time (5 years), the progression of the disease was very slow. This prompted us to perform bulk transcriptome analyses from serially collected blood samples to study the expression of the genes involved during this natural history Figure 1lgorithm  to eluciFunctional enrichment performed with time course transcriptome expression profile  highly active against cancer cells such as melanoma . Catheps-IS quantification analyses during TKI interruption period. IL-21 has been shown to participate to the NK cell expansion of K562 cell line engineered to express membrane-bound IL-21 feeder in combination with IL-2  followed the BCR::ABLith IL-2 , 23.S1P  receptorOverall, the molecular analyses reported during the long-term TKI discontinuation in this patient allowed to detect major modulation of genes involved with NK cell activity. Interestingly, several genes that we identified to be part of the immunological pathway seemed to be activated with a successful TFR after TKI discontinuation , 27–29. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by INSERM ethical committee. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the patients/participants for the publication of this case report.JI, PM, NO and DC: Performed molecular Single cell experiments; CD: Bioinformatics analyses; XF performed BCR-ABL quantification experiments, JF, AB-G: validated BCR-ABL data, AT, AB-G: Clinical follow-up, plan experiments, JI, PM, CD, AB-G, AT: wrote the paper. All authors contributed to the article and approved the submitted version


Antimicrobial resistance (AMR) is an increasingly severe threat to global public health that requires action across different sectors. Selection of appropriate antimicrobials is an urgent challenge due to the emergence of drug resistance. In 2017, Kenya developed an AMR policy and National Action Plan to drive prevention and containment of AMR. A priority activity under AMR surveillance strategic objective was to develop a national AMR training curriculum for in-service healthcare workers. In this paper we discuss the development process, gains achieved through implementation across the country and lessons learned.An initial stakeholders’ forum was convened to brainstorm on the process for developing the curriculum and some issues deliberated upon include the design approach, development roadmap, curriculum outline and scope, delivery, and evaluation methodologies. A dedicated team of subject matter experts (SMEs), drawn from the project and government ministries, compiled the initial draft of the curriculum and later the training materials. A series of other stakeholders’ meetings were convened to review these materials. The National Antimicrobial Stewardship Interagency Committee (NASIC) of the MOH in Kenya identified a team of experts from academia, research, and government to work with the SMEs in reviewing and providing valuable inputs to the curriculum. Additionally, principles of adult learning and a One Health approach for development were considered as AMR has drivers and impacts across sectors. A validation workshop was held to finalize the documents with a formal launch conducted during the World Antibiotics Awareness Week of 2020.A multisectoral AMR surveillance training curriculum and facilitator and trainee manuals were developed and endorsed by MOH and Ministry of Agriculture, Livestock, Fisheries and Cooperatives within one year. Over 500 healthcare workers in 19 counties were trained, with overwhelming adoption by other stakeholders in Kenya and beyond.This curriculum was developed to standardize training for AMR detection and surveillance. The central role played by the MOH ensured expeditious development and roll-out of this curriculum. The in-service curriculum, now available on an e-learning platform, provides a ready opportunity to build capacity of healthcare professionals. Additional resources are needed to standardize and scale these efforts to reach all healthcare workers. Antimicrobial resistance (AMR) is a growing global public health threat requiring concerted efforts across human, animal, and environmental health sectors . AMR occIn recognition of the growing threat of AMR, in 2015 the World Health Organization developed a Global Action Plan on AMR following a 2014 resolution on AMR at the World Health Assembly (WHA 67.25), which called on the agency to develop a draft global action plan to ensure that all countries have the capacity to combat AMR . In 2017AMR surveillance is one of the strategic objectives of the Kenya National Action Plan. Other objectives include the need to increase knowledge and awareness of AMR, antimicrobial stewardship, infection prevention and control, and research and development. The Joint External Evaluation conducted in 2017 using the International Health Regulations (2005) benchmarks revealed that Kenya had made progress in strengthening its capacity to prevent, detect, report, and respond to public health threats, but significant gaps remained under the AMR core capacity. AMR detection, surveillance and antimicrobial stewardship indicators each had a low score of two (2) out of the possible score of five (5) , 2017. AThe second strategic objective in the Kenya National Action Plan is to strengthen the awareness and the evidence base for AMR . KnowledAMR surveillance in Kenya, like in many other developing countries, is still in its nascent stages, with limited coverage . The devUSAID’s Infectious Disease Detection and Surveillance (IDDS) project is a global health security project strengthening infectious diseases detection and surveillance systems in more than 20 countries, including Kenya. In Kenya, the project works closely with NASIC within the MOH to combat AMR nationally. In this article we discuss the technical assistance provided to the Government of Kenya in the development of an in-service AMR surveillance curriculum and training materials, roll-out, and implementation.The need for an in-service AMR surveillance training curriculum was recognized by stakeholders prior to the start of concerted efforts to combat AMR in the country in 2017 . This waA stakeholders’ forum was convened by NASIC with the support of the USAID IDDS project. An expert from academia made presentations on the curriculum development process. Stakeholders brainstormed several aspects including the curriculum outline and scope, delivery, and evaluation methodologies, and agreed that the new materials would not replace pre-service curricula. Stakeholders emphasized the need to adopt a One Health approach in the development process because AMR drivers are interdisciplinary .NASIC convened all stakeholders’ meetings during the development process for review of materials with technical assistance from IDDS. A team of subject matter experts (SMEs) from IDDS, MOH, Ministry of Agriculture, Livestock, Fisheries and Cooperatives, and key academic institutions worked on curriculum outlines, content outlines, intended learner outcomes, training content, evaluations, and a Monitoring & Evaluation plan for the curriculum. These materials were built on the recommendations from the initial stakeholders’ workshop. The team developed participant and facilitator manuals. The SMEs adopted a peer-to-peer review approach where one peer reviewed materials developed by another. The materials were submitted to an in-country panel of experts identified by NASIC. After their reviews, the materials were submitted to external reviewers and editors. A validation workshop was convened for stakeholders to confirm that the curriculum and its related materials captured the national aspiration to fill knowledge and practice gaps among in-service healthcare professionals. Later in the year, the documents were officially launched by MOH and Ministry of Agriculture, Livestock, Fisheries and Cooperatives. The curriculum was designed to have objectives at two levels: for participants completing pre-analytic modules and participants completing analytical and post-analytic modules. The pre-analytic modules aim to equip participants with knowledge and skills to:i) Develop and deploy customized antimicrobial use and AMR containment and prevention interventions;ii) Apply a One Health approach in addressing AMR agenda; andiii) Coordinate, monitor, and evaluate multisectoral stewardship activities targeted at mitigating AMR.The analytic and post-analytic modules of the course were designed to equip learners with knowledge and skills to:i) Utilize advances in diagnostic techniques  and design methods to incorporate their use/application to stewardship initiatives;ii) Perform AMR surveillance monitoring, evaluation, and reporting; andiii) Use antimicrobials judiciously to minimize or eliminate the occurrence of AMR.The recommended course duration for laboratory practitioners is five days, due to the hands-on microbiology techniques module, while three days are recommended for other healthcare cadres. This modular approach can be adapted to respond to specific needs of the target audience. The curriculum mainly targets in-service healthcare workers who play various roles in AMR diagnostics and surveillance across animal and human health sectors. These include clinicians, veterinarians, veterinary paraprofessionals, laboratory personnel, pharmacists, nurses, biomedical engineers, informatics, and records officers. Preliminary modules are designed to raise awareness of AMR, and thus can be offered to leaders for AMR resource mobilization and allocation, especially at the national and county levels.Mixed delivery methods were proposed based on principles of adult learning . DeliverThe curriculum is organized into modules, each focusing on a specific area based on: (a) the need to share information on AMR, or (b) the need to transfer knowledge and skills on AMR diagnosis and surveillance. All modules have multiple choice questions to check participants’ knowledge before and after the training. Both trainer and participant course evaluation forms were developed to capture valuable feedback for improvement of the curriculum and materials. Data collected through these evaluations and other informal feedback mechanisms would be used to assess effectiveness, impact, and inform improvements to the curriculum and course content during the first review cycle (two years after the launch).A validation workshop was convened on January 8, 2020, and attended by 63 AMR stakeholders from multiple sectors; these national and sub-national stakeholders reviewed and approved the curriculum and its associated training materials for publication and implementation.After finalization of the curriculum and training materials, IDDS provided support to NASIC to organize a five-day “training of trainers” session covering the 12 curriculum modules. The training convened participants from human and animal sectors to support roll-out of the curriculum. These included 18 laboratory practitioners, 3 nurses, 3 monitoring and evaluation professionals, 2 pharmacists, 1 public health officer, 1 medical officer, and 2 veterinary officers. Scores increased from a pre-test average of 61% to a post-test average of 87%. This training also served as a pilot through which valuable inputs were provided by both trainers and participants that were helpful with improving the materials. Key feedback guided revision of content scope against time allocated. Some modules content, such as biosafety and biosecurity, were reduced to keep the focus on AMR while more time was allocated to other modules, including the clinical guide.In 2020, IDDS, in collaboration with five counties and NASIC, conducted a two-day AMR surveillance sensitization training based on the new curriculum, to create awareness among different stakeholders of AMR and ensure a robust response to the threat of AMR. The objectives of the training were to improve health workers’ awareness and understanding of AMR surveillance in the counties, build capacity on best practices to adopt and promote appropriate use of antibiotics and avert AMR, and how to conduct AMR surveillance in the counties aligned with Kenya policy guidelines on AMR surveillance.The target audience for the training were clinicians, veterinarians, veterinary paraprofessionals, laboratory personnel, pharmacists, nurses, public health officers, records officers, and health policy– and decision-making authorities.A total of nine of the 12 modules in the AMR surveillance training curriculum were delivered. These included foundational modules that built awareness of AMR, appropriate use of antimicrobial agents; infection prevention and control; drivers of AMR at the human-animal-environment interface; diagnostic stewardship; specimen collection, transport, reception, and storage; specimen referral and reporting; procurement and supply chain management; clinical guide; and prudent use of antimicrobials in veterinary practice. These sessions were facilitated by the individuals trained during the “training of trainer” sessions. Due to the heterogeneity of the participants, pre- and post-training assessments were not administered, however post training feedback was obtained using a structured checklist that evaluated how the participants would use the knowledge acquired, potential barriers to use of the acquired knowledge and overall rating of the training. There was an above-average rating of the training offered across the five counties with recommendations to increase duration of the training or tailor sessions to a particular area each time. A total of 180 healthcare professionals were reached. Distribution is shown in A hands-on training workshop for medical laboratory technologists was organized that included some of the modules in the AMR surveillance curriculum. Modules giving overview of biosafety and biosecurity measures, pre-analytical processes related to specimen collection, transportation to the laboratory, reception, and storage, laboratory diagnostic processes related to organism identification, antimicrobial susceptibility testing, and results reporting, specimen/isolate referral system, supplies quantification, procurement systems, equipment management processes and quality assurance (QA) in a microbiology laboratory were covered. A total of 15 participants were trained. Participants knowledge and attitudes on AMR and AMR detection were evaluated before and after the training. Pre- and post- training scores were 57.6 and 72.8%, respectively.The Kenya AMR surveillance curriculum was developed and finalized in a period of less than a year. This is in contrast to the usual curriculum development timelines that can span years . At the Also, in less than 2 years after finalization of the curriculum and the training materials, over 500 healthcare workers have been trained or sensitized using these materials in human sector from 19 of the 47 Kenyan counties. The curriculum materials were quickly adopted by other stakeholders in the country supporting AMR surveillance and used to build capacity in the counties they support. Key among these are partners in Fleming Fund consortium and global health projects funded by the U.S. Centers for Disease Control and Prevention in Kenya. International collaboration has raised awareness of AMR as a global health challenge and spurred actions within the counties to address the threat. Sensitization of clinicians has promoted utilization of bacteriology services, though the volume of requests still falls below targets. While many healthcare workers in the human health sector have been reached, there is need to scale capacity building of healthcare workers in the animal health sector. One-time sensitization forums may not be adequate to drive the desired behavior change in use of antimicrobials among healthcare professionals. Globally, irrational use of antimicrobials has been escalating over several decades ; thus, tThe Kenya AMR surveillance curriculum and related training materials are set to standardize capacity building initiatives and facilitate building of competencies required in healthcare professionals for the fight against AMR in Kenya. While the curriculum has been well received, additional investments are needed to reach a tangible representation of healthcare workers in both the human and animal health sectors in the country. A dedicated, multisectoral team of SMEs with government ownership and support of curriculum development experts and professional reviewers and editors can accelerate Kenya’s progress toward AMR prevention and mitigation.The original contributions presented in the study are included in the article/JN, JO, SC, DM, and MN primarily created the manuscript with review and input from the AD, AT, ET, EW, RN, RG, SG, and WM. DM handled the project administration. All authors contributed to the article and approved the submitted version.This work was made possible through the support of the United States Agency for International Development (USAID), Global Health under the terms of the Infectious Disease Detection and Surveillance contract GS00Q14OADU119. Views expressed are not necessarily those of USAID or the United States government.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.