I recount some landmark discoveries that initially confirmed the cyclic AMP response element-binding (CREB) protein-memory consolidation and allocation linkages. This work constitutes one of the successes of the field of Molecular and Cellular Cognition (MCC) but is also of interest to philosophers of neuroscience. Two approaches, “mechanism” and “ruthless reductionism”, claim to account for this case, yet these accounts differ in one crucial way. I explain this difference and argue that both the experiment designs and discussions of these discoveries by MCC scientists better fit the ruthless reductionist's account. This conclusion leads to further philosophical discussion about how discoveries in cellular/molecular neurobiology integrate with systems neuroscience findings. And if they didn't, they should. Because the more we advance our knowledge in molecular neurobiology, the more we realize that CREB plays a pivotal role in the response of neurons to external stimuli” 3−) to various sites on the inactive protein. Phosphorylated CREB dimerizes with other CREB binding proteins to affect gene transcription. Some phosphorylated CREB isoforms“CREB” is the acronym for cyclic adenosine monophosphate (cAMP) response element-binding protein, a gene transcription factor that binds to specific DNA sequences  on the control region of various genes to influence their expression. CREB is activated by phosphorylation, the attachment of a phosphate group  and cAMP; and that CREB functioned as a substrate for depolarization-activated calmodulin kinase II (CaMKII) and protein kinase A Could a role for CREB be demonstrated in mammalian memory consolidation? Circumstantial evidence suggested that might be possible. Michael Greenberg's laboratory had established that CREB was activated as a gene transcription factor in mammalian neurons by two intracellular second messengers, calcium ions . In one of those remarkably serendipitous coincidences on which so many episodes of scientific progress turn, Günther Schütz, a molecular biologist from Heidelberg, Germany, was attending symposia at CSHL to present his research on the role of CREB in liver function. Schütz's lab had developed a homozygous CREB α/δ-isoform knock-out mouse. They provided mutants and wild-type littermate controls to Silva's lab for neurophysiological and behavioral testing.Using Schütz's mice, a team in Silva's lab led by Roussoudan Bourtchouladze showed intact early phase, but deficient late phase LTP in mutant hippocampus slices, as compared to wild-type littermate control mice CREB's basic molecular biology was likewise becoming clarified. Besides the g-protein-coupled serotonin/dopamine-responding receptors → adenylyl cyclase → cAMP cascade, at least three other intracellular signaling pathways were shown to initiate CREB phosphorylation and activation as a transcription factor.These were exactly the results that Abel et al. found Combining all of these initial experimental results, Abel et al. sketch “a framework for a molecular understanding of the consolidation of explicit long-term memory” +) influx into post-synaptic neurons, generating excitatory post-synaptic potentials, and in turn action potentials (“spikes”). The result of increased AMPA receptors at synaptic sites is specific post-synaptic neurons that generate stronger excitatory post-synaptic potentials, and hence are more likely to generate action potentials, to later excitatory neurotransmitter release from the same pre-synaptic cells, for extended periods of time, with this synaptic plasticity now linked to performance on behavioral measures of memory consolidation. As yet, however, no evidence exists that these AMPA receptor movements require CREB transcription.While there is still no consensus as to what are the critical downstream effectors of CREB activation for LTP or memory consolidation, evidence has been found for the movement of α-amino-3-hydroxl-5-methyl-4-isoxazoproprionic acid (AMPA) receptors from both intracellular storage pools and extrasynaptic sites to synaptic sites in the postsynaptic density. AMPA receptors, activated by the neurotransmitter glutamate, are the principal channels for sodium ion , in mouse LA. GFP expression, coupled in these mice to CREB transgene expression, causes neurons in tissue slices to glow green under standard light microscopy. She trained mutant mice and wild-type littermate controls on amygdala-dependent tone-fear conditioning. Images from GFP slices were compared, neuron for neuron with images from ARC fluorescent slices, to see how many neurons overlapped, i.e., had both high CREB activity driven by the transgene insertion at time of learning and high recent activity during the retrieval phase of a specific tone-shock conditioning revealed by in situ hybridization of nucleus ARC mRNAs. CREB-enhanced cells were three to four times more likely than non-infected neurons to show ARC expression, indicating high activity during specific memory retrieval. In contrast, decreasing the levels of CREB in neurons via virus insertion of a mutated CREB gene that interfered with CREB function had the opposite effect. Cells transfected with mutant CREB were only one-twelfth as likely to express nucleus ARC mRNA at retrieval time than non-infected neurons. Numerous control experiments were necessary to interpret these findings, but in the end Han et al. conclude “our results suggest a competitive model of memory formation, in which eligible neurons are selected to participate in a memory trace as a function of their relative CREB activity at the time of learning” This role for high CREB activation in the allocation of specific tone-fear memories was further elaborated by subsequent studies. One example from Silva's lab comes from a team led by Yu Zhou, who attached an engineered gene from Drosophila for an allatostatin receptor, insertable and expressible in mammalian neurons Another follow-up study came from Josselyn's lab. Jin-Hee Han led a team that instead killed the transgenic CREB-enhanced LA neurons after their preferential recruitment into a specific tone-shock memory Even this brief history of the CREB-memory linkage elaborates Dudai's 3.Two contrasting accounts in recent philosophy of neuroscience, “mechanism” and “ruthless reductionism”, both claim to incorporate findings such as the CREB-memory consolidation and allocation linkages. Not surprisingly, these two approaches agree about much concerning neuroscience's practices and products. Both accounts propose to start philosophical reflection from actual neuroscience practices, and so both recognize that philosophical assertions made about neuroscience must answer to what actually happens in labs and gets published in papers. Both accounts recognize the centrality of manipulation or intervention experiments, and both recognize a related variety of distinct types of such experiments. Both accounts view neuroscience as focused primarily, or at least centrally on discovering mechanisms. Both acknowledge explicitly that the work of cellular and molecular neuroscientists makes important contributions to explaining cognitive phenomena. This last point of agreement is especially important because it rejects the tendency of many other philosophers to focus exclusively on cognitive and systems neuroscience as the only fields in the discipline that are of philosophical relevance. Both mechanists and ruthless reductionists insist that such a limited focus, though popular, leads to a grossly distorted picture of neuroscience as a scientific endeavor.of the next level up, e.g., of the individual hippocampus neurons undergoing LTP. And the latter is the mechanism of the next level up, and so on, until finally the penultimate level from the top is the mechanism for the system's behavior, e.g., the rat navigating the water maze. The ruthless reductionist, on the other hand, is perfectly willing to claim that the molecular mechanisms are a mechanism of the system's behavior, so long as the right collection of integrated intervention experiments confirm this.These points of agreement are found throughout the most cited writings of mechanists not mechanisms of the organism's (system's) behavior. Instead, they are mechanisms of functioning individual neurons, conducting action potentials and transmitting activity to other neurons across shared chemical synapses. Individual neurons in turn are mechanisms of functioning neural circuitries or networks, and on up the mechanism hierarchy ultimately to the behaving system. According to Bechtel, by locating each mechanism at its proper level in the nested hierarchy, the mechanist account does not falsely accord the molecular mechanisms any special epistemic or explanatory role or privilege. They are just another level of mechanisms hierarchically organized to constitute the cognitive system. Bechtel insists that this recognition accords to mechanist philosophy of neuroscience an epistemic advantage over ruthless reductionist: “What is gained by paying attention to each level of organization identified in the course of decomposition is an understanding of the variety of roles that must be performed in producing the overall phenomena” For those who wonder whether this distinction actually constitutes a difference, consider that mechanists make a big deal over it. According to Bechtel the mechanist account fully accommodates molecular neurobiology, but also locates those mechanisms at their appropriate level in the hierarchy of mechanisms-within-mechanisms constituting the cognitive system Ignoring intermediate levels of mechanisms-within-mechanisms and advocating for direct mind-to-molecular pathways linkages reminds Bechtel of a similar “ruthlessly reductive” approach that in retrospect wreaked havoc in genetics: The legacy of focusing on genes as responsible for traits, ignoring the variety of factors involved in the regulation and expression of genes, illustrates the risks involved. Researchers often claimed success in explaining biological traits as soon as they identified a responsible gene, failing to recognize that often many factors, including other genes as well as environmental processes, are required to generate the trait relevant for the specific cognitive operation under investigation. On the other hand, Craver insists, pursuing ruthlessly reductionist neuroscience, and thus ignoring top-down constraints imposed on lower level investigations by fields of neuroscience that address higher level components in the nested hierarchy, runs serious risk of losing sight of the boundaries of the mechanisms for specific cognitive functions, and thereby including in one's explanation more processes than just the relevant ones. In this context Craver quotes with approval philosopher Phillip Kitcher's famous quip about reductionism (ruthless or otherwise): “a world viewed only at the fundamental level would be a world of gory details unfiltered by a higher level perspective” Another leading mechanist, Carl Craver, argues that recognizing the nested hierarchies of mechanisms-within-mechanisms permits mechanists to identify the specific components, activities, and organization that are Since both mechanists and ruthless reductionists agree that philosophy should “start with neuroscience,” it is fair to ask, how did the neuroscientists who forged the CREB-memory consolidation and allocation linkages (i) design their experiments and (ii) describe their results? Did they do so more in keeping with the mechanists' stress on the proper place of molecular mechanisms within a nested hierarchy, or more in keeping with the ruthless reductionists' stress on finding direct linkages of cognitive functions to molecular processes? Do their experiments and their discussions look more like One source of data for answering such questions are scientists' discussions in landmark experimental publications. Careful attention to these discussions is part of a method that Silva, Landreth and Bickle call “metascience”  on the overall behaving system (organism) are monitored. Intermediate levels of nested mechanisms-within-mechanisms are not direct targets of the experimental manipulations or interventions. Of course, the experimental manipulations and interventions of these molecular pathways will have measurable effects on intermediate-level mechanisms. If manipulating CREB or PKA molecules in specific neurons produces effects on the organism's behavior, doing so will also produce effects in specific neural circuits and regions, and on the plasticity of the individual neurons that compose them. That is a simple fact of componency organization. But in the research sketched in the previous section, those intermediate-level effects were not the ultimate targets of the researchers. Typically those researchers did measure cell-physiological effects of their interventions, such as LTP in the specific neurons they intervened into. But these measurements were just a step on the way to their ultimate aim, namely, of showing that manipulation of a single component of a known molecular causal pathway in vivo had measurable effects on behavioral indicators for a specific cognitive function, e.g., ones for explicit memory in rodents. In reference to system (organism) behavior at the top level. That is what Bourtchouladze et al. showed first with Schütz's CREB knock-out mice Without question there are neuroscientists who design experiments, construct computational models, and discuss their results in language reflecting mechanists' image of nested hierarchies of mechanisms-within-mechanisms. Bechtel presents numerous examples molecular mechanisms of cognitive functions, without appealing to any stepwise restriction of mechanisms only to the next layer up in some nested hierarchy. In their original CaMKII mutation study, Silva, Tonegawa and colleagues took their in vivo behavioral data as indicating “that α-CaMKII has a prominent role in spatial learning” a mutation in a known gene is linked to a specific mammalian learning deficit, and indicates that single genetic changes can have a selective but drastic impact on learning and memory” the gene/molecule experimentally intervened upon affected the cognitive function itself, operationalized behaviorally for experimental testing. One can interpret these claims along mechanistic lines, by insisting that these scientists were simply leaving the hierarchy of intervening intermediate-level mechanisms unstated, perhaps as enthymemes. But doing this requires putting additional words in these scientists' published writings. The scientists themselves don't include those additional words. So recognizing the defeasibility of metascientific conclusions mentioned above, ruthless reductionism seems better to capture what these scientists both did and wrote about what they did.Furthermore, these scientists speak explicitly of having discovered CREB-dependent transcription in mammalian long-term memory” CREB as an important component of memory consolidation in mammals” of the cognitive function itself, just as the ruthless reductionist pictures it . What successful experiments of the sort surveyed in section 1 do is link controlled experimental manipulations of a component of the molecular pathways directly to behavioral effects, obviating the st century scientific image still rest on a metaphor grounded in theological disputes from more than eight centuries ago?The “levels” metaphor is so ingrained in both science and philosophy of science that its demise is probably too much to hope for. But a better metaphor would be one of concentric organism-shaped blobs of coarser and finer grain, each one taking up the entire space of the behaving organism. The entire behaving system would be the concentric blob of coarsest grain, the networks or circuits of neurons the concentric next-finer blob, the entire collection of individual neurons comprising all these networks or circuits the next finer, and the entire collection of intra- and inter-neuronal molecular pathways the next finer still. No “ontological levels” require bridging on this picture; one and the same system is organized at coarser and finer grains, and our experimental technologies enable us to manipulate single components in the molecular pathways in order to evoke significant alterations in system (organism) behavior. Ironically, the “levels” metaphor, along with many of its current puzzles, trace back to medieval “levels of Being” discussions. Must our early-21From a metascientific perspective, the scientists who initially linked CREB activity to memory consolidation and allocation designed their experiments and described their results more like ruthless reductionists than like mechanists. So ruthless reductionism is alive and well in the practices of quite a few neuroscientists. Mechanism's popularity in recent philosophy of neuroscience does not reflect a dominance in actual neuroscience-in-practice. But these philosophical-metascientific reflections raise other concerns. What implications might these differences in experimental practices and explanatory goals carry for unification and integration of results across the numerous disciplines that compose contemporary neuroscience?4.I have argued that some landmark results from the first twenty years of MCC reflects ruthlessly reductive neuroscience practice. I have also assumed that other neuroscientists, including some MCC researchers, pursue work more in the fashion championed by mechanists. Ruthless reductionism and mechanism differ in at least one significant respect, as depicted in On the contrary. Why should neuroscientists have to speak in one voice about how to integrate results across fields? Neuroscience is an inherently interdisciplinary science. Neuroscientists come from a variety of scientific backgrounds, ranging from physics to chemistry to molecular biology, up to systems biology, experimental psychology, and cognitive science. The scientific intuitions and hunches about how best to address a given phenomenon will differ as widely as do neuroscientists' backgrounds and interests. Why expect “big picture” unity among such a motley crew, or hope that one philosophical approach will reflect the work of everybody involved in the enterprise? Instead, “why can't we all just get along”, and pursue whichever problems strike us as important and addressable, using whichever tools and experiment designs strike us as most promising, and executing experiments and discussing results in whatever way strikes us as most appropriate given our myriad scientific heritages? And simply accept that different neuroscientists will make different choices at each point?neuroscience was employed indeed reflects the project of ruthless reductionism, a narrowing of focus to the molecular processes within individual neurons” Bechtel suggests something like this happily disunified picture and mechanistic. And no doubt a whole lot else: computationalist, evolutionary, dynamicist, extended/enactive... And there's no reason for it not to be all these things. “Highly interdisciplinary” implies highly variable, in experimental methods, research tools, designs, goals, explanatory practices, assumptions... In short, in all the ways that make up a given scientist's practices or “approach.” The philosophy of such a field should reflect this variance.If Bechtel's historical narrative is accurate—and I have no reason to doubt that it is—then from a metascientific perspective contemporary neuroscience is both ruthlessly reductive The Journal of Neuroscience, currently divides each issue into five distinct areas, with different editors and reviewers for each, and authors typically choose the appropriate area for their submission: cellular/molecular; development/plasticity/repair; systems/circuits; behavioral/cognitive; and neurobiology of disease (http://www.jneurosci.org/). So despite the field's overall variety of approaches, neuroscience “on the hoof” maintains rather sharp and institutionalized divisions. A variety of flora blooms across the entire field, but that should not lead one to think that it is all one big, happy cross-fertilizing garden. At least in their day-to-day lab work, most neuroscientists spend time with others who are closely aligned with their own training and interests. This feature of actual neuroscience practice is a topic worth serious philosophical reflection. Perhaps “multidisciplinary” rather than “interdisciplinary” is a more apt descriptor for contemporary (and foreseeable?) “neuroscience-in-practice”? How much do these real-world restrictions limit our capacity to integrate neuroscientific results that span the molecular to the cognitive—a goal that to this day remains much more as happy rhetoric than concrete accomplishment? These reflections beg for further philosophical-cum-scientific investigations.However, it is also important to realize that no field this varied can be tension-free. “Interdisciplinary” does not mean or imply “ecumenically cooperative”. In fact, “interdisciplinary” as understood by administrators—the picture of scientists from these various disciplines constantly mixing and matching up in joint research endeavors and adopting the tools and approaches of their fellows—is relatively rare. The physical locations of differently focused neuroscientists often differ. Cognitive and systems neuroscientists are often housed on academic campuses; cellular and molecular neuroscientists are often housed on medical campuses. Cognitive and systems neuroscientists in the United States often apply for research funding to the National Science Foundation and private research sources; cellular and molecular neuroscientists more often turn to the National Institutes for Health. In research projects involving neuroscientists from across these divides, as for example in drug discovery and development, the specific tasks of each participant are typically highly circumscribed and limited to their own areas of expertise. The Society for Neuroscience's official journal,


Anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) are systemic necrotizing vasculitides associated with significant morbidity and mortality. Given the immunosuppression used to manage these conditions, it is important for clinicians to recognize complications, especially infectious ones, which may arise during treatment. Kaposi sarcoma (KS) is a lymphoangioproliferative neoplasm caused by human herpes virus 8 (HHV-8). Its cutaneous manifestations can mimic vasculitis. We describe a 77-year-old man with microscopic polyangiitis with pulmonary-renal syndrome treated with prednisone and intravenous cyclophosphamide who developed KS (HHV-8 positive) after 2 months of treatment. Cyclophosphamide was discontinued and prednisone gradually lowered with improvement and clinical stabilization of KS lesions. This comprehensive review includes all published cases of KS in patients with AAV, with a goal to summarize potential risk factors including the clinical characteristics of vasculitis, treatment and outcomes of patients with this rare complication of immunosuppressive therapy. We also expanded our literature review to KS in other forms of systemic vasculitis. Our case-based review emphasizes the importance of considering infectious complications of immunosuppressive therapy, especially glucocorticoids, and highlights the rare association of KS in systemic vasculitis. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) includes microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA) (Wegener’s), and eosinophilic granulomatosis with polyangiitis (EGPA) (Churg-Strauss syndrome), all pauci-immune vasculitides which share clinical features and are characterized by the presence of ANCA . Serious2 during hospitalization. He was able to successfully come off hemodialysis after 1.5 weeks with a new baseline creatinine of 2 mg/dL. Four weeks later, he was treated with cycle 2 of intravenous cyclophosphamide 400 mg/m2.A 77-year-old man of Italian American descent was referred to us for evaluation of AAV. He presented to an outside facility with a 30-lb weight loss, cough, scant hemoptysis, and worsening dyspnea on exertion. Laboratory evaluation showed acute kidney injury with a creatinine of 2.58 mg/dL baseline 1.0 mg/dL) and BUN of 30 mg/dL, acute anemia with hemoglobin 6.1 g/dL. During hospitalization, he developed rapidly progressive renal failure requiring initiation of hemodialysis, in addition to gross hemoptysis. Serologies included a positive anti-nuclear antibody (ANA 1:320), positive double-stranded DNA (dsDNA 1:40), p-ANCA of 1:1280, MPO 52 IU (> 1 IU positive), and negative anti-glomerular basement membrane antibody (Table .0 mg/dL 2) infusion had been administered 2 weeks prior. Apart from the rash, he denied any symptoms. Laboratory parameters, including renal function, were stable. Medication review revealed that he had been on treatment with hydralazine for hypertension for more than 1 year prior, and, given association of hydralazine with AAV, hydralazine was discontinued. Prednisone was lowered to 35 mg. Rituximab was discussed given severe manifestations of vasculitis but given positive hepatitis B core antibody, recommendation was made for evaluation with infectious diseases first. Unfortunately, 1 month later, he was hospitalized for mental status changes from urosepsis with Escherichia coli bacteremia. He was on prednisone 35 mg daily at the time. Treatment was complicated with Clostridium difficile colitis. During that hospitalization, further testing was pursued (Table Approximately 2 months after starting treatment, he developed a new lower extremity rash Fig. a. PredniWe present a rare case of MPA, possibly hydralazine-induced, with pulmonary-renal syndrome complicated by the development of KS during treatment with systemic glucocorticoids and cyclophosphamide.One of the unusual aspects of this case was the possibility of the manifestations of AAV being hydralazine-induced. Severe manifestations including pulmonary-renal syndrome have been described in hydralazine-induced vasculitis –12. A clThe risk factors for KS include infection with HHV8, HIV, and immunosuppression –6, 13–15Clostridium difficile requiring hospitalizations which delayed initiation of rituximab. Finally, he also developed infectious complication of KS from HHV-8. Given that there was no evidence of active vasculitis, that hydralazine which may have been a trigger was discontinued, and risks of further immunosuppression, decision was made to use glucocorticoid monotherapy, lower prednisone gradually monitor closely. He clinically improved with reduction in glucocorticoid doses, discontinuation of cyclophosphamide, and topical imiquimod. To date, there has been no recurrence of vasculitis which may be in part from the discontinuation of hydralazine.A review of the literature evaluating KS in AAV, identified 10 additional cases whose findings are summarized in Table Given the rarity of KS in AAV, we also extended our literature review to other forms of systemic vasculitis Table . We founBoth the cellular and humoral arms of the immune system have been implicated in the control of KS. Immunosuppression is a common theme noted in KS, whether due to innate problems of host immunity, or, due to factors that lead to induced immunosuppression . For insFinally, B-cells are latent reservoirs of HHV-8 . While aIn summary, despite immunosuppression in vasculitis, KS appears to be a rare complication of therapy. It is important to recognize KS as an infectious complication in patients with AAV. The violaceous, nodular lesions in KS, can be mistaken for the palpable purpura from cutaneous vasculitis which also affect the upper and lower extremities . The maj


A similar trend was seen in total CT score . The level of agreement between ReDS and CT changes yielded a perfect result. Statistically significant changes were observed in lactate dehydrogenase, lymphocytes, and c-reactive protein over 2 weeks. This pilot study shows that ReDS can track changes in lung involvement according to the severity of COVID-19. Further studies to detect early clinical deterioration are needed.Remote dielectric sensing (ReDS) is a non-invasive electromagnetic wave technology which provides an accurate reading of lung fluid content, and it has been reported as a valid tool in monitoring heart failure patients. Considering that morphological alterations in COVID-19 include pulmonary edema, the purpose of the present study was to evaluate the reliability of ReDS technology in assessing the excess of lung fluid status in COVID-19 pneumonia, as compared to CT scans. In this pilot single center study, confirmed COVID-19 patients were enrolled on admission to an intermediate care unit. Measurements with the ReDS system and CT scans were performed on admission and at weeks 1 and 2. Eleven patients were recruited. The average change in ReDS was −3.1 ± 1.7 after one week ( In December of 2019, an outbreak of a novel coronavirus disease (COVID-19) occurred in Wuhan, a city in the Chinese province of Hubei, and, thereafter, it dramatically spread worldwide ,4,5,6. CChest computed tomography (CT) is crucial to assess the pattern as well as extent of lung lesions, and the most common radiological pictures include pure “ground-glass” opacities (GGO), GGO with reticular and/or interlobular septal thickening, GGO with consolidations, crazy paving, and pure consolidations ,14,15. AA further non-invasive device, intended to measure the dielectric properties of tissues, mainly determined by fluid content, is electromagnetic energy-based technology, named remote dielectric sensing . In detail, ReDS™ is an FDA- and CE-cleared device that measures lung fluid content quickly, in absolute terms, providing objective and reproducible indices of volume status ,24,25,26Considering that morphological alterations in COVID-19 include pulmonary edema, the purpose of the current pilot study was to evaluate the reliability of ReDS technology in assessing the excess of lung fluid status in COVID-19 pneumonia, as compared to CT scans, and to explore its potential role in monitoring the clinical evolution of these patients by measuring the correlations with longitudinal changes of radiological features and selected serological parameters.The ReDS™ Pro System is a point of care device, consisting of two sensors in a clip configuration, that is applied on the patient for 45 s long measurement. When applying the device, the sensors are positioned on the front and back of the patient’s thorax with no need for direct skin contact, allowing measurements to be performed through light clothing A. The deLow-power electromagnetic signals are transmitted through the thorax between the sensors, and the intercepted signals reflect the dielectric properties of different tissues. Since lung tissue is primarily composed of water and air, with water having a very high dielectric coefficient and air having the lowest dielectric coefficient, the average dielectric coefficient reflects the percentage of lung tissue fluid content. The normal value of lung fluid volume is between 20% and 35%. The full range of lung fluid volume reported by the ReDS system spans from 15% to 60%.The ReDS technology has FDA and CE approvals, and it is indicated for assessment of fluid overload. Currently, it is being used for the measurement and monitoring of lung fluid content in heart failure patients ,10,11,13The study protocol was approved by the local ethical committee  and performed at the University Hospital “Ospedali Riuniti”, Ancona, Italy.Consecutive patients, aged ≥ 18 years, admitted to the sub-intensive care unit between 10 April (first day of availability of ReDS device) and 4 May 2020  with a diagnosis of pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), confirmed by reverse transcription polymerase chain reaction (RT-PCR) performed on a naso-pharyngeal swab or bronchoalveolar lavage, were prospectively enrolled in the present study according to eligibility criteria. Exclusion criteria were thorax deformity, recent (last 3 months) chest trauma or rib fractures, presence of a pacemaker, inability to remain seated during measurements, and inability to give an informed consent.2/FiO2)P/F), white blood cells count (WBC), lymphocytes count (Ly), d-dimer, interleukin 6 (IL-6), C-reactive protein (CRP), lactate dehydrogenase (LDH), B-type natriuretic peptide (BNP), and need for oxygen support, including non-invasive mechanical ventilation (nIMV). nIMV was indicated with SpO2 < 92% on oxygen therapy 15 L/min Fi O2 50%, while mechanical ventilation (IMV) was indicated when respiratory rate was above 25/min and/or signs of acute respiratory failure despite nIMV. The follow-up period was the time from admission to the date of discharge or death. Main clinical outcomes  were collected during the follow-up period.Demographic factors and selected clinical characteristics were collected for all cases. These included: age, sex, smoking history, height, weight, comorbidities, gas exchange values , at 7 days (t1), and at 15 days (t2) later. Each measurement was performed by trained operators (F.M. and A.D.M.B.) according to standard procedure.All patients underwent chest CT scans, combining high resolution (HRCT) and pulmonary angiogram protocol (CTPA), on admission and 2 weeks later. Each CT scan was evaluated independently and by two pulmonologists with 10 and 12 years of experience in order to define the main radiological pattern and total extent of parenchymal involvement. Final decisions were reached by consensus. Each CT evaluation by reporting pulmonologists was blind to the ReDS data.The prevalent morphological patterns at the CT scan were classified as follows: 0 = pure GGO; 1 = crazy paving; 2 = GGO with consolidations; 3 = consolidations; 4 = consolidations with interseptal thickening.The extent of parenchymal involvement at CT was scored by visually evaluating the percentage of lesions’ involvement at the lobar basis according to a 5-point categorical scale, using the following scoring system: 0 = none (0%), 1 = minimal (1–25%), 2 = mild (26–50%), 3 = moderate (51–75%), 4 = severe (76–100%). The total severity score (TSS) was reached by summing the five lobe scores (range from 0 to 20); severe parenchymal lung involvement was defined as a TSS value of 8 or more .t-test, and correlation methods. Statistical significance was declared when the p-value was found to be less than or equal to 0.05 (two sided). All statistical analyses were performed using the SAS statistical software or equivalent statistical software.The primary endpoint was the concordance between CT scores and ReDS values. The secondary endpoint was the concordance between ReDS and laboratory results. The parameters’ values and their differences were compared using descriptive statistics, 2. Comorbidities were present in more than two thirds of patients (72.7%) and blood hypertension was the most prevalent condition, whereas none reported a pre-existing cardiac disease. The mean value of P/F was 152.8 ± 70.1. All patients received oxygen support and three of them required nIMV (27.7%). D-dimer, LDH, and CRP were altered in most of the study cohort , while BNP was normal for all patients .A total of eleven patients were included in the present study. Demographic and clinical characteristics at baseline  for all patients are summarized in Chest CT scans revealed diffuse pulmonary alterations in all patients and most of them (81.8%) presented with severe involvement (TSS ≥ 8), with a mean TSS at baseline of 10.1. Consolidations only (36.4%) and ground glass opacities plus consolidations (36.4%) were the most prevalent morphological pattern, followed by crazy paving and pure GGO . No further abnormalities were observed, except for a mild unilateral pleural effusion in one patient.The mean value of ReDS measurements at baseline was 29.6 ± 7.6%, without significant difference between left and right lung. ReDS measurements greater than the upper limit of normal  in at least one lung were present in 5 patients (45%).p = 0.001) and 4.6 ± 2.9  after two weeks (p = 0.001) (2), fraction of inspired oxygen (FiO2) and their ratio (P/F) were also significant  B. The lenificant , Table 2The present study firstly describes a novel approach to assess and monitor lung fluid excess in patients hospitalized for COVID-19 pneumonia using ReDS technology. Our findings overall show a not negligible proportion of patients with fluid overload in at least one lung, and a significant longitudinal change of ReDS readings over time, which were strongly correlated with CT score and selected clinical features. Notably, ReDS measurements corresponded to clinical evolution throughout hospitalization, supporting the potential utility of ReDS in monitoring COVID-19 pneumonia.Pulmonary fluid overload during COVID-19 pneumonia is thought to be the expression of lung edema due to hyper-inflammation, one of the leading pathological mechanisms causing respiratory failure during the first “exudative phase” . This isThe excellent correlation between ReDS readings and CT-measured lung water was already established in previous studies. In particular, Amir et al. demonstrated a high correlation between the ReDS system and CT results in patients with heart failure  and theyA major advantage of the ReDS™ system is that it is safe, non-invasive, non-ionizing, easy to use, and requires minimal patient collaboration. Moreover, it is available at the bedside, leading to reducing the contagious risk for health-workers during patient transportation and the need to sanitize larger areas of equipment . Italy was the first Western country to experience this unexpected and devastating pandemic, with a multitude of infected patients admitted to emergency departments over few weeks. This led to an urgent and profound re-organization of the national healthcare system, including the widespread adoption of telemedicine and remote counselling ,32, as wMajor strengths of this pilot study include the novelty of results, the prospective nature of the study design that determined an accurate collection of data for each patient, and the objective measurements of the main outcome.The study has some limitations. First, the clinical study was strictly observational, by study design, and therefore the role of the ReDS™ system in improving outcomes could not be determined. Second, the choice of a cutoff point may be criticized due to a lack of prospective validation. We chose to use this cutoff according to prior published works suggesting that normal lung water content is 20–35%. However, the main purpose of this pilot study was not to determine the proportion of COVID-19 patients with significant fluid excess, but to assess role of this device as a monitoring tool, evaluating how ReDS values  correlated to CT features and to clinical evolution. A third limitation is the limited number of patients included.To our knowledge, this is the first pilot study that aimed to measure lung fluid overload in COVID-19 pneumonia with ReDS technology, suggest that ReDS technology has potential for monitoring these patients at bedside and avoiding repeated CT scans for longitudinal evaluations.Larger, multicenter studies are needed to confirm these findings, to determine the appropriate timing for longitudinal evaluation, and to explore the clinical impact of the ReDS™ system on the management of patients hospitalized with COVID-19 pneumonia.


Mycobacterium bovis is a causal agent of bovine tuberculosis (bTB), one of the most important diseases currently facing the cattle industry worldwide. Tracing the source of M. bovis infections of livestock is an important tool for understanding the epidemiology of bTB and defining control/eradication strategies. In this study, whole genome sequencing (WGS) of 74 M. bovis isolates sourced from naturally infected cattle in the State of Rio Grande do Sul (RS), southern Brazil, was used to evaluate the population structure of M. bovis in the region, identify potential transmission events and date the introduction of clonal complex (CC) European 2 (Eu2). In silico spoligotyping identified 11 distinct patterns including four new profiles and two CCs, European 1 (Eu1) and Eu2. The analyses revealed a high level of genetic diversity in the majority of herds and identified putative transmission clusters that suggested that within- and between-herd transmission is occurring in RS. In addition, a comparison with other published M. bovis isolates from Argentina, Brazil, Paraguay and Uruguay demonstrated some evidence for a possible cross-border transmission of CC Eu1 into RS from Uruguay or Argentina. An estimated date for the introduction of CC Eu2 into RS in the middle of the 19th century correlated with the historical introduction of cattle into RS to improve existing local breeds. These findings contribute to the understanding of the population structure of M. bovis in southern Brazil and highlight the potential of WGS in surveillance and helping to identify bTB transmission. Supplementary files 1 and 2 (available in the online version of this article).Raw sequencing data for all sequenced isolates have been deposited in the European Nucleotide Archive (ENA) under project PRJEB39667. Accession numbers are detailed in File S1.https://github.com/avantonder/Brazil2020.The R code used to analyse the data in this paper is available on Github: Mycobacterium bovis is a causal agent of bovine tuberculosis (bTB), one of the most important diseases currently facing the cattle industry worldwide. Tracing the source of M. bovis infections of livestock is an important tool for understanding the epidemiology of bTB and defining control/eradication strategies. This is, to date, the largest whole genome sequencing-based study of M. bovis in Brazil and South America and will be of interest to many researchers in the bacterial genomics field. Our study highlighted the diversity of M. bovis in southern Brazil, identified potential within- and between-herd transmission often over large distances, and estimated a potential date of introduction of clonal complex Eu2 into the region that correlates with the historical introduction of cattle into the State of Rio Grande do Sul to improve existing local breeds. Further, the work showed that even with limited sampling, genome sequencing has the potential to be a useful tool for helping to control bTB transmission in Brazil.Mycobacterium bovis is a causative agent of bovine tuberculosis (bTB), infecting mainly bovines globally, in addition to a broad host range that includes humans and wild animals. Moreover, the disease in humans constitutes a public health problem and currently is one of the most important diseases faced by the global cattle industry, causing significant economic losses to beef and dairy production  and of the American isolates is 1968 [95 % CI: 1961–1974; The state of RS was originally located in Spanish territory, to the west of the area covered by the Treaty of Tordesillas. The first Europeans to colonize the state were Jesuit priests, who founded missions and imported cattle from the Iberian Peninsula where Eu2 is the predominant genotype . After tM. bovis in southern Brazil. Even with the limited sampling, we were able to detect within- and between-herd transmission at different SNP thresholds and found supporting evidence for one of these transmission events in the form of known cattle movements between farms with the same owner (herds C and D). Future studies could focus on collecting a larger number isolates from more herds with a focus on adjacent farms where the cattle movement data are available and bTB prevalence is consistently high.A limitation of this study was the small sample size compared to the number of infected cattle in the region, estimated to be in the region of 84 000 head in 2013. Although this was due to limited available resources, clearly this hampered the detection of transmission events. However, we were still able to characterize significant diversity within the herds sampled and provide the first WGS-based description of the population structure of Recently, the Brazilian bTB control programme (PNCEBT)  strategyClick here for additional data file.Click here for additional data file.


Dear Editor,1 The D1-like receptors, comprising DRD1 and DRD5, primarily couple to the Gs family of G proteins to activate adenylyl cyclase and induce cAMP production. DRD1 is the most abundantly expressed dopamine receptor in the CNS.1 It is the central receptor mediating excitatory dopamine signaling in multiple dopaminergic pathways. Dysregulation of DRD1 signaling has been directly linked to Parkinson’s disease (PD), schizophrenia, and drug abuse.2 Due to its fundamental functions in human diseases, DRD1 has long been the subject of intensive drug development efforts toward the treatment of neuropsychiatric diseases.3 A majority of DRD1 agonists, including the SKF compounds, targets the orthosteric pocket of DRD1, but none has passed clinical trials for neuropsychiatric symptoms to date.3Dopamine acts as an essential neurotransmitter whose signaling is conducted through five G protein-coupled receptors (GPCRs), dopamine D1 to D5 receptors (DRD1–DRD5).4 Multiple groups have reported that DRD1 PAMs, such as LY3154207, CID2886111, and DETQ, stimulate DRD1 signaling.6 With ongoing clinical investigation, DRD1 PAMs may offer new therapeutic opportunities for PD.7GPCR positive allosteric modulators (PAMs) have been proposed to provide unique advantages over orthosteric agonists including greater receptor subtype selectivity, saturable therapeutic effects and the ability to maintain spatial and temporal patterns of endogenous dopamine signaling, which collectively may lead to reduced side effects.s complexes activated by the endogenous ligand, dopamine, and a synthetic agonist, SKF81297, both in the presence of LY3154207, respectively  to assemble DRD1–Gs signaling complexes8  human DRD1, miniGαs_DN, rat Gβ1 and bovine Gγ2 in Sf9 insect cells. The complexes were prepared as described in 6 were added to further stabilize the dopamine-bound DRD1–Gs complex. The structures of DRD1–Gs complexed with dopamine/LY3154207 and SKF81297/LY3154207 were determined at a global resolution of 3.2 Å and 3.0 Å, respectively  value of 0.6 Å for the main chain Cα atoms. However, the orthosteric binding pocket (OBP) of SKF81297 is narrower compared to that of dopamine Fig. . In both3.32 , which is highly conserved in aminergic GPCRs.9 Such interaction is further enhanced by hydrogen bond interactions among D1033.32, S1073.36, and W3217.43  Fig. . The pri.43 Fig. . The catTM6 Fig. . These fing Fig. . For SKFing Fig. . As a re3.32, S1985.42 and N2926.55 largely decreased the potency of dopamine  for a β2AR PAM named Cmpd-6FA11  and the interactions between DRD1 and SKF81297 are nearly identical, but the binding poses of SKF81297 are slightly different and conformational differences can be observed in the allosteric binding pocket (Fig. para-hydroxyl group in the SKF81297 catechol ring and S1985.42 (Fig. s-coupling12. It is likely that LY3154207 potentiates the orthosteric agonist-induced DRD1 signaling by stabilizing the active state of DRD1, which is similar to the allosteric modulation of β2AR by Cmpd-6FA.11We have also determined a cryo-EM structure of DRD1–Gket Fig. . In the .42 Fig. , and theIn conclusion, the two structures reported here uncovered the unique binding modes of dopamine and the DRD1 potent PAM LY3154207, revealed the detailed mechanism of how DRD1 is occupied by both endogenous and synthetic agonists with different chemical scaffolds, and elucidated the potential allosteric regulation of DRD1 activity. Structural comparison demonstrated the important role of the DRD1 EBP in determining its ligand potency. In addition, the structure of LY3154207 bound to DRD1 presents a first view of the DRD1 PAM-binding pocket. Together with mutagenesis results, our structures provide a framework for more efficient and subtype-selective ligand discovery targeting DRD1 for treating CNS diseases, both at the orthosteric ligand pocket and at the allosteric pocket.Supplementary information


Unfortunately, less than one-third  of the respondents had only heard about antimicrobial stewardship. Participants showed a positive attitude toward prudent antimicrobial use and were open to solutions to the AMR crisis. We noticed a certain gap between farm veterinarians’ desire to improve and perform better in daily practice, while at the same time feeling like they did not have enough guidance, help, and resources.Antimicrobial resistance (AMR) is considered one of the most prevalent global health issues in both veterinarian and human medicine. This complex problem requires a “One Health” approach with the cooperation of all healthcare sectors, as well as agriculture, finance, and consumers. We conducted a survey with the objective to assess the knowledge and attitudes of farm animal veterinarians toward AMR and antimicrobial use in the Republic of Serbia with a small focus on mastitis therapy. A total of 110 respondents completed the questionnaire, which represents a response rate of 27.3%. The majority of our respondents ( Antimicrobial resistance (AMR) is a global health threat that continues to rise in both veterinary and human medicine . The cauExtensive research has been done in human medicine regarding the misuse of antimicrobials ,17, whilIt is very important to obtain a better understanding of veterinarians’ prescribing habits since they prescribe antimicrobials for prophylactic, metaphylactic, or therapeutic purposes . Even thAMS describes all strategies and actions that can help the optimization and the rational use of antimicrobials . Since tFarm animals are exposed to considerable quantities of antimicrobials , especiaHowever, the concerns about AMR require the dairy industry to reduce the use of antibiotics. Since, in the Republic of Serbia, there are no developed guidelines for the rational AMU in veterinary medicine, there is an urgent need for understanding the attitude and knowledge of veterinarians toward primarily AMR, but also AMU and AMS, as a crucial step for the design of strategies and interventions to combat this public health threat . FurtherIn the Republic of Serbia, total AMU in human medicine was found to be well above the European average ,30, whilConsidering all the factors mentioned, and the fact that, to our knowledge, there have not been similar articles published, the objective of this study was to assess attitudes and knowledge regarding AMR and AMU among farm animal veterinarians in Serbia.n = 92, 83.6%), while females made up 16.4% (n = 18) of respondents. There were 25.5% of respondents who were 25–34 years old and only one participant (0.1%) over 65 years old. Participants were of different educational levels, with 76 (69.1%) having a first degree, six (5.5%) having a master’s degree, 14 (12.7%) having a doctorate, and 14 (12.7%) having a specialist degree. Eighty-four respondents (76.4%) worked in the private sector, while 23.6% worked in the state sector. Of the 110 veterinarians, more than half had already worked in practice for 6–15 years  and one-third  had more than 15 years of work experience.A total of 110 farm animal veterinarians participated in the survey. A summary of sociodemographic data is presented in n = 80, 72.7%) had received some sort of educational classes regarding AMU and/or AMR in the last 3 years, while 29 of them (26.4%) had no education on the subject at all, and one participant (0.9%) had no recollection of either happening. Many respondents used foreign or domestic guidelines when prescribing antibiotic therapy with 32 (29.1%) using them often, 39 (35.5%) using them moderately, and 25 (22.7%) using them rarely. Only six (5.5%) participants did not use the guidelines, while eight (7.2%) considered that there were no good quality guidelines available. Even so, 97 (88.2%) respondents thought that there was a need for more local guidelines, and only seven (6.4%) thought that there was no need for them.A great majority of respondents , weekly , or monthly  practice, while 44 (40%) of them experienced those situations rarely and two (1.8%) experienced them never  of respondents indicated that they had protocols for prescribing antibiotics in their practice, while 80% (n = 88) did not. However, 87.3% (n = 96) considered that those protocols should exist, while 12.7% (n = 14) disagreed. A majority  kept records of AMU, and some  did not. Regarding prescribing antibiotics outside of suggested clinical indications for their usage, respondents did it often , moderately , rarely , or never . When asked to which extent the use of antibiotics by unqualified people negatively impacts AMR, 91.8% (n = 101) of participants considered the impact to be significant, 7.3% (n = 8) considered it moderate, and 0.9% (n = 1) thought that there was no impact  of respondents knew what the term meant, while more than two-thirds  had never heard about it (n = 21) of respondents thought that the implementation of AMS would not lead to any significant changes, while the rest assumed that it would lead to a reduction in AMR in humans and animals and an increase in the responsible use of antibiotics  and contact with animals  to be the main sources of AMR in humans , followed by “rational prescribing of AB”  and “farm hygiene”   .n = 87, 20.5%) and vaccines , and least familiar with phage therapy  and nanoparticles   .n = 103, 93.6%), “AB use without clear indications” , “wrong therapy length” , and “low dosage of AB”  had a great impact on this issue  and the lack of quick diagnostic tests , followed by pressure from animal owners , prescribing habits , the lack of clear guidelines for certain diseases , and insufficient education of veterinarians   .n = 78, 70.9%), antibiograms , and milk withholding period for drugs  as very important. The spread of AMR amongst people and animals also had a high status with 51.8% (n = 57) and 56.4% (n = 62) of respondents, respectively, rating them as very important  prescribed antibiotics exclusively for therapeutic purposes  to the data describing respondents’ opinions on rationally prescribing antibiotics shows that the first two dimensions (Ds) described more than 30% of data inertia . The position of recorded answers in the space defined by D1 and D2 revealed a separative grouping, mostly in the space defined by D2. Specifically, we can notice that respondents that considered AMR an important factor when prescribing antibiotics also never prescribed them without clear indication —negative part of D2. Furthermore, in the negative part of D1, respondents that did not take AMR into consideration and did not think that handling of antibiotics by unqualified people could be detrimental for AMR were separately grouped, in addition to veterinarians that often prescribed antibiotics outside their indications and did not have prescribing protocols or collected data on AMU . The posn = 99, 90.0%) that it will become an even bigger problem in the near future if we do not act rapidly. Slightly less than half of the respondents  strongly agreed that they contributed to the spread of AMR, and 85 (77.3%) of them were completely open to new solutions in the forms of alternatives to antibiotics if clinically proven to be effective. The respondents also had strong opinions on over-the-counter antibiotics, with 79.1% (n = 87) of them completely agreeing it should be a prohibited practice (Participants acknowledged that AMR is an emerging problem in both human and veterinary medicine today with 102 (92.7%) completely agreeing, no participants completely disagreeing, and only two (1.8%) slightly disagreeing with this. They also mostly completely agreed . The position of the recorded answers in the space defined by D1 and D2 highlights the grouping of veterinarians that considered AMR a big issue currently and even worse in the near future, and that performed antibiograms routinely—in the negative part of D1 and positive part of D2. Furthermore, the same respondents shared the opinion of prohibiting over-the-counter use of antibiotics and also considered that the uncontrolled use of antibiotics on farms is important for AMR in humans, but did not think that we have sufficient data on the transmission of AMR from farm animals to humans. On the other hand, respondents that considered AMR exclusively a hospital setting issue did not perform antibiograms routinely and were localized in the negative part of D1, as well as the negative part of D2. Moreover, they did not consider that using antibiotics without prescription is dangerous, or that uncontrolled use of antimicrobials on farms can also increase AMR in humans .n = 56, 17.4%) and amoxicillin , followed by amoxicillin + clavulanic acid . The least used antibiotics were erythromycin , lincomycin , and novobiocin   .n = 91, 60.6%). Furthermore, only 16.7% (n = 25) of respondents used an antibiogram when prescribing antibiotics in therapy  were 35–44 years old, with 84.5% having at least 6 years of experience working in the practice.As mentioned before, AMR represents a global health threat, and a large number of organizations are working on decreasing drug resistance worldwide. One established method to combat AMR is the implementation of good practice AMU guidelines ,36. As iUnfortunately, less than one-third of our participants (30.9%) were previously familiar with the term “antimicrobial stewardship”, which shows that there is still a lot of work to be done in promoting this concept and raising awareness in the veterinary sector to facilitate the implementation of AMS strategies. This can be explained by it being a relatively new term in the veterinary profession, and veterinarians might be familiar with its principles only in theory. Contrary to our findings, 63% of veterinarians from Nigeria were familiar with the AMS term , while, Regrettably, AMS programs are yet to be widely applied in veterinary medicine. In line with the global WHO action plan , the RepA very low percentage (20%) of our respondents had protocols for prescribing antibiotics in their practices compared to more than half of Nigerian respondents  and moreRegarding the strong correlation between AMU and the spread and development of AMR , it is iA study conducted in the Netherlands suggested that veterinarians with a positive attitude and sufficient knowledge of AMR can have a positive impact on AMU . HoweverWhen it comes to alternatives to antibiotics, the veterinarians in our study were most familiar with probiotics (20.5%) and vaccines (18.9%), while phage therapy (2.1%) and nanoparticles (1.7%) were less known to them. Our results are similar to a study where vaccines for the prevention and control of calf scours were recommended on 24% of dairy farms in Italy . ContrarA systematic review article of 34 studies published in 2021 showed that the most important factors influencing veterinary health professionals’ selection of an antibiotic in therapy are sociodemographic characteristics, influenced by different attitudes, business factors, and complacency, as well as owner-related factors, such as lack of awareness and demand for antibiotics . AnotherOur findings also suggest a correlation between veterinarians that perform antibiogram tests routinely and the awareness level of how uncontrolled AMU influences AMR development. Forty-nine percent of our respondents performed antibiograms routinely, which is a higher level compared to the 38% of European veterinarians  and loweEven though the respondents primarily prescribed antibiotics for therapeutic purposes, there was still a presence of prophylactic and metaphylactic use of these drugs that may have consequences for the increase in AMR . SimilarAlmost all of the surveyed veterinarians (92.7%) completely agreed that AMR is an important issue in both human and veterinary medicine, corresponding with most Bhutan veterinarians (96%) . FurtherWithin the livestock sector, one of the biggest threats to animal health and welfare, which is also considered as the most common disease that causes huge economic losses in the dairy industry, is bovine mastitis . The etiCertain limitations can be attributed to questionnaire-based studies. Their subjective nature depends on the participants’ opinions and memory. There is also a risk of misinterpretation of questions, which we tried to reduce with as many closed questions as possible. The recorded response rate was 27.3%, which is comparable to similar studies ,87. Our Ethical review and approval of this questionnaire were granted by the University of Novi Sad, Faculty of Agriculture Ethics Committee, via an ethics review application .According to the data obtained from the Veterinary Chamber of the Republic of Serbia, it is estimated that there are around 700 licensed farm animal veterinarians . The questionnaire was distributed via the Association of Veterinarians Practitioners of Serbia (AVPS) to their 403 registered members that are currently working in large or mixed veterinary practices, since not all licensed veterinarians are working in practice with farm animals. The questionnaire was distributed using the Google Forms platform. The survey link was sent via emails to the members of the AVPS. The survey was available online for 3 months between 14 May and 14 August 2021. A total of 110 respondents completed the questionnaire, resulting in a response rate of 27.3%.The questionnaire used in the study was designed with the intention to collect data on the knowledge and attitudes of farm animal veterinarians on the subjects of AMR and AMU during the period May to August 2021.The survey was developed after a comprehensive review of the related literature and consultation among the members of research team. The questionnaire was created using a combination of original questions and questions from various surveys ,37,44,70The full questionnaire is provided in The questionnaire consisted of 28 questions across four sections aimed at collecting data on demographics, as well as knowledge and attitudes of farm animal veterinarians toward AMR, AMU, and AMS, with a section focused on mastitis therapy. The four sections were designated as sociodemographic data, significance of antimicrobial resistance, rational use of antibiotics, and knowledge of antimicrobial resistance. The first section consisted of six questions regarding demographic data, level of education, and career choices, including age, gender, workplace, the highest level of education, years in practice and average monthly workload based on the number of patients. The second section with 10 questions was focused on participants’ views on the significance of AMR and AMS. The goal was to assess whether the participants kept up to date with this issue and their attitudes toward it in their daily practice. The third section had eight questions about participants’ prescribing habits, factors influencing them, and the rational use of antimicrobials. In the fourth section, the respondents were presented with eight statements with five different levels of agreement . The statements were used to assess the attitudes and knowledge of participants regarding AMR. Additionally, this section had questions about the most used antibiotics in cow mastitis therapy and the factors influencing decision making when prescribing this therapy.The results were extracted from Google Forms to Microsoft Excel. Data were checked for errors, consistency, and uniformity. Statsoft Statistica 12.5  was used to process the data by means of univariate and multivariate statistical methods. Specifically, the obtained responses were analyzed by descriptive statistics and analysis of response frequency. Furthermore, multivariate correspondence analysis (MCA) was performed in order to better evaluate the complexity of the data matrix structure.MCA is specially designed for the evaluation of relationships among categorical variables, whether nominal or ordinal. It is a dimension reduction technique, thus enabling data analysts to have better insight into model variability by graphical representation of evaluated variables in the space defined by lower number of dimensions . The calculated dimensions describe a lower proportion of dataset variability (often termed as inertia), but reveal a better structure of the dataset model. In this research, MCA was applied to the dataset represented by a matrix with dimensions of 110 × 75, thus containing a total of 8250 inputs .The assessed attitudes and knowledge of Serbian farm veterinarians toward AMR and AMU highlight their mostly reflective attitudes, acknowledging the issue as very important. Veterinarians are trying to educate themselves on AMR; however, there are certain gaps in their knowledge, mainly regarding AMR etiology, personal responsibility when prescribing antibiotics in therapy, use of antibiotics in prophylactic and metaphylactic therapies, and prescribing antibiotics without clear indication (AST tests). The latter, however, seems to be influenced by factors somewhat out of veterinarians’ control, i.e., mainly the cost and the lack of available tests. Most veterinarians also realized the effect that AMU and rational prescription have on the AMR issue, and they were willing to use AMU guidelines to help them in their everyday work, making guidelines for prudent AMU in cattle production systems extremely needed. There was dissatisfaction regarding the number of available local guidelines and the use of antibiotics by unqualified people. Moreover, veterinarians had very positive attitudes toward the possibilities of alternatives to antibiotics.Unfortunately, the respondents showed a substandard understanding of AMS, making further education and promotion of AMS concepts one of the priorities in our fight for the reduction in AMU and AMR. Additional surveys should be conducted to continuously check the status of our findings.


We suspect that the pyramids are initiated by Ag co-sputtered onto the surface. Growth of pyramids is due to the gathering of step edges with (16 × 2) reconstruction around the pyramid base during layer-by-layer removal of the substrate, and conversion to {19 13 1} faceting. The absence of pyramids using Ar+ energies above 400 eV is likely due to surface damage that is insufficiently annealed.Isolated pyramids, 30–80 nm wide and 3–20 nm tall, form during sputter-annealing cycles on the Ge (110) surface. Pyramids have four walls with {19 13 1} faceting and a steep mound at the apex. We used scanning tunneling microscopy (STM) under ultrahigh vacuum conditions to periodically image the surface at ion energies between 100 eV and 500 eV and incremental total flux. Pyramids are seen using Ar Surface features formed through ion bombardment depend on many parameters, including sputter ion type, fluence, flux, energy, and incident angle; sample type, orientation, and temperature; as well as sample cleanliness and the presence and type of contaminants and defects. The methods of formation reported in the literature are varied, including dot and ripple patterns , facetedThis paper discusses the formation of isolated pyramids with {19 13 1} faceted sides that form during sputter-annealing cleaning cycles performed on the Ge 110) surface. Interesting optical and biological applications for pyramids such as these are discussed in other works [ surface.Each cleaning cycle is 15 min of ion bombardment with the sample at 500–650 °C, followed by 10 min of annealing the sample to 800 °C. Usually, this process results in a Ge (110) surface with large, atomically flat domains of surface reconstructions—ideal for imaging with scanning tunneling microscopy (STM). Pyramids are only observed when performing cleaning cycles on two types of samples: Ge (110) dosed with 10 monolayers (MLs) of Ag, and bare Ge (110) using a sample holder that must still have had many MLs of Ag. In the absence of Ag, the surface becomes smooth from sputter-annealing, and no pyramid formation is observed . Thus, w17 to 1 × 1020 cm−2, showing that a sample becomes very rough at high fluences, but the surfaces in this experiment showed little change throughout the fluence range of 4.5 × 1016 to 1.8 × 1018 cm−2.Many effects are often associated with ion bombardment, such as collision cascade, sputtering, surface damage, ion implantation, amorphization of the surface, viscous flow, and ion reflection and re-deposition. Theories about pattern formation during ion bombardment at high temperatures typically discuss an interplay between roughening (due to bombardment and anisotropic surface diffusion) and smoothing (due to surface diffusion processes) ,13,14,15Simultaneous co-deposition of even very small amounts of metal during ion bombardment has been found to initiate different formations during ion bombardment, such as cones and whiskers ,18,19,20We used STM under ultrahigh vacuum (UHV) conditions to periodically image the surface at different ion energies and fluences. The evolution of the surface suggests that the pyramids are nucleated by Ag clusters co-sputtered onto the surface, and that the pyramids grow due to layer-by-layer removal of the substrate near the pyramid base.2 square samples were manually cut with a diamond scribe, rinsed in methanol and then hydrogen peroxide, before placing them into the STM–LEEM sample holder prior to insertion into the UHV chamber with a base pressure of + ions with energies ranging from 100 to 500 eV at an incident angle of 34° from the direction normal to the surface, with the sample continuing to cool during the ion bombardment. New samples were used for experiments with different bombardment energies. We imaged the sample every 5–7 cleaning cycles, and the samples went through a total of ~40 sputter-annealing cycles. The sputtering current could vary between 2 and 8 μA, depending on the energy, partial pressure of Ar, and the sample distance from the gun. Typical sputtering parameters for these measurements were Ar+ ion partial pressure of 13 s−1 cm−2. The fluence ranged from 4.5 × 1016 to 1.8 × 1018 cm−2, but since we varied our ion energy between 100 and 500 eV, the fluence range varied for different ion energies. The direction of sputtering is incident from the right side when observing the STM images in this work. The top plate of the sample holder was machined from Mo , an STM , and an X-ray photoemission spectrometer  . Ge (110Examples of pyramid formations on the Ge (110) surface are shown in Positive surface features such as these pyramids are expected to be stable under ion bombardment due to reduction of sputtering at local topographical maxima and enhanced sputtering at local minima ,25,26,27In contrast to the stability of pyramids, step edges change location during the sputter-annealing process. On a gradually stepped surface without these pyramids, our sputter-annealing parameters would typically cause step edges to recede in the direction of the higher terrace due to anisotropic surface diffusion combined with layer-by-layer removal of the substrate ,28,29,30We suspect that the pyramids are immobile upon nucleation, and their locations inhibit the movement of step edges; this results in bunching-up of step edges, and the formation of local {17 15 1} faceting. While the four walls of the pyramids are typically similar in size, the {17 15 1} faceting generally forms long sections perpendicular to the sputtering direction . Additionally, the faceting forms along paths bordering pyramids. In many cases, the faceting wraps closely around pyramids and groups of pyramids, and examples of this are present on the right half and the bottom of + after 14, 23, and 33 cleaning cycles. After 14 cleaning cycles, the surface is still rough on an atomic scale, with each small dot in ep edges .When the sample is bombarded with 200 eV ions a–e, isol17 cm−2 to 1.7 × 1018 cm−2) for any given ion energy. The surface changes the most with lower numbers of cleaning cycles, because new samples are rough, as shown in In general, Once the surface forms large surface domains, which typically takes 8–16 cleaning cycles, additional cleaning cycles cause fewer changes to the surface. This is in contrast with other ion bombardment experiments, where increasing fluence caused an increase in surface roughness ,15,16,32The step density of the substrate was higher on the sample that underwent more cleaning cycles. The location of steps near the pyramids supports two hypotheses: (1) pyramids inhibit local terrace removal, and (2) a component of pyramid growth is through pinning of step edges at the pyramid’s base. Even though the step edges form {17 15 1} faceting when further from the pyramid, they must convert to {19 13 1} in order to contribute to pyramid growth.Pyramids are often clustered together, suggesting that pyramid locations may encourage the initiation of others nearby. We suspect that pyramids inhibit the local rate of terrace removal and, thus, cause the bunching of steps; nearby locations at the top of bunched-up steps might be ideal for the initiation of new pyramids. Contaminant clusters that initiate pyramids at these locations may experience quicker growth, since sputtering of nearby step edges can quickly contribute to their height. This may enhance the stability of newly formed pyramids against disintegration.Scanning artifacts are present in many of the STM images, where the scanning tip often “jumped” near the apex. This was common on pyramids formed when Ag was co-sputtered from the sample holder, but pyramids formed following sputter-cleaning of Ag-dosed samples were more often imaged clearly. This may be related to the steep slope, roughness, contamination, or disorganization of the peaks.+ ions. This figure shows a progression similar to that seen in + energies between 200 eV and 400 eV caused four-sided pyramids to form, similar to those already discussed. The pyramids formed with 400 eV Ar+ were often larger than pyramids formed with lower energy Ar+ and had larger mounds at the apex. Similarly, the substrate sputtered with 400 eV Ar+ had longer inclines of densely packed step edges. The slopes of the walls of larger pyramids were sometimes different along one direction compared with the rest of the pyramid  and makes their height greater than it would be without this effect.+ ions and the coincidental creation of Frenkel pairs near the surface. Positive growth through anisotropic adatom migration was found to be the primary method of growth for similar pyramids [Another effect of ion bombardment that should be addressed is the implantation of Arpyramids . Adatomspyramids . Due to pyramids ,20,28,29The reconstruction on {19 13 1} consists of rows of atomic clusters. The short-range order of clusters runs along the + between 200 eV and 400 eV and require Ag to be present on the sample or sample holder. The sputter-annealing cycles cause step edges to be pinned around the base of existing pyramids, while surface layers are locally removed in a layer-by-layer fashion. The inhibition of the migration of step edges at the perimeter of the pyramids is proposed to be the primary component of pyramid growth. As each terrace is removed around the location of the pyramid, the reconstruction at the base of the pyramid converts to {19 13 1} faceting, and the pyramid grows in height by one atomic layer. The absence of pyramids using 500 eV Ar+ is suspected to be due to surface damage that is insufficiently removed during the annealing cycles. The surface reconstruction of the {19 13 1} faceting displays bumps associated with atomic clusters similar to those on the {17 15 1} faceting, and a model is provided.Isolated pyramids form during sputter-annealing cycles on the Ge (110) surface. Pyramids have four walls with {19 13 1} faceting and a steep mound at the apex. Pyramids form using Ar


They produced high inorganic electrical voltage coefficients, high electromechanical coupling coefficients, and environmentally friendly properties that enhance the electromechanical performance of pure PVDF nanofibers, and they are all the critical requirements for modern flexible pressure sensors. In detail, PVDF and PVDF-based composites nanofibers were prepared by electrospinning, and different flexible sandwich composite devices were fabricated by the PDMS encapsulation method. As a result, the six-time enhancement maximum output voltage was obtained in a PVDF-BNBT (fiber)-based composite sensor compared to the pure PVDF one. Our results indicate that the output voltage of the pressure sensors has been significantly enhanced, and the development gate is enabled by analyzing the related physical process and influence mechanism.Wearable energy harvesters and sensors have recently attracted significant attention with the rapid development of artificial intelligence and the Internet of Things (IoT). Compared to high-output bulk materials, these wearable devices are mainly fabricated by thin-film-based materials that limit their application. Therefore, the enhancement of output voltage and power for these devices has recently become an urgent topic. In this paper, the lead-free bismuth titanate-barium titanate (0.93(Na In recent years, microelectromechanical systems (MEMS) devices, energy harvesters, and flexible sensors have received rapid development based on the extensive growth and enhanced performance of the piezoelectric nanostructures ,2,3,4,5.Nowadays, polymer materials, especially PVDF and PVDF copolymers with high sensitivity and deformability, are considered as the most promising materials, which can meet the requirements of the flexibility characteristics and the dynamic tactile sensing of wearable electronic devices products ,9,10. Ind) of PVDF, there were still many improvements for developing a commence piezoelectric PVDF device. In addition, research indicated that doping was an effective way to improve the piezoelectric properties of PVDF, which was critical to further enhance the piezoelectric sensor performance [3) nanocomposite to prepare high-performance flexible piezoelectric nanogenerators, which produced a higher maximum output voltage of 13.2 V [Compared with bulk film materials, one-dimensional ferroelectric materials with ultra-high specific surface area and excellent piezoelectric properties showed good application prospects in piezoelectric devices ,13. To sformance ,20. Yu eformance . Chen etf 13.2 V .d33 ~500 pC/N [Considering the perovskite inorganic piezoelectric materials having higher piezoelectric and dielectric constants, such as barium titanate (BTO), lead zirconate titanate (PZT), lead magnesium niobate-lead titanate (PMN-PT) ,22, etc.500 pC/N . TherefoThe BNBT particles and BNBT nanofibers were prepared based on a sol-gel process . For thew/w) mixture at a PVDF powder/solvent weight ratio of 1:9. Then, the electrospinning was conducted at 25 kV, 15 cm spacing between needle tip and collector, and a feed rate of 1.5 mL/h. The as-spun nanofibers were dried at 80 °C for 4 h. For the PVDF-BNBT (particle) and PVDF-BNBT (nanofiber) composite nanofibers fabrication, before the electrospinning process, the BNBT particles and BNBT nanofiber (30 wt% of the PVDF powder weight) were first dispersed into the PVDF spinning solution. Then, the electrospinning was conducted under the same conditions as the pure PVDF.For the PVDF spinning solution, the raw PVDF powder was dissolved in the N, N-Dimethylformamide , and acetone   under Cu Kα radiation. A field emission scanning electron microscope  was used to characterize the fiber samples and to determine the diameters of the fibers. The secondary electron image mode was adopted. Both the as-spun nanofibers and annealed samples were coated with gold. Fourier transform infrared (FTIR) spectra of the fibers were measured using a Thermo fisher Nicolet iS10 system .The BNBT particles were obtained from BNBT precursor solution, dried in an oven at about 80 °C to volatilize the solution method, and then annealed at 750 °C . Figure β phase can be detected after combining BNBT particles and BNBT nanofibers.2, and the thickness was 20 μm. Under the same pressing and releasing process provided by the manual test stand force testing crimper (the applied mass is 100 g), the output signals from each sensor were directly measured by oscilloscope and shown in 2) and can create a local electric field. This electric field will induce the nearby PVDF molecule bond mechanical stretching, thereby improving the piezoelectric phase content of the PVDF, accompanying the improvement of the piezoelectric performance. On the other hand, it is found that the PVDF-BNBT (fiber) nanofiber-based sensor produced a better performance than another PVDF-BNBT (particle) nanofiber-based sensor. This result may be due to a larger and longer size of BNBT fibers attached to the surface of PVDF nanofiber, resulting in better local polarization stretching and resulting stress of PVDF-BNBT (fiber) composite nanofiber. Therefore, enhanced piezoelectric and electromechanical responses were obtained.Furthermore, to give an insight into the electromechanical performance of different composite nanofibers, three flexible pressure sensors based on PVDF nanofibers, PVDF-BNBT (particle) composite nanofibers, and PVDF-BNBT (fiber) composite nanofibers, respectively, were fabricated as sandwich structures, as shown in In summary, PVDF-BNBT composite nanofibers were investigated, and it was found that the three nanofibers , and PVDF-BNBT (fiber)) had similar diameter, which indicated the embedded process did not change the physical dimension of the nanofiber. An advantage of the enhanced direct piezoelectric effect of the PVDF-BNBT (fiber) composite nanofibers was demonstrated in the application of piezoelectric pressure sensors. In application, three flexible sensors can produce the RMS output voltage of 1.1 V (pure PVDF nanofibers), 2 V (PVDF-BNBT (particle) composite nanofibers), and 3.57 V (PVDF-BNBT (fiber) composite nanofibers), respectively, under the same vibration excitation. It is found that the highest output voltage was obtained in PVDF-BNBT (fiber) composite nanofiber-based piezoelectric sensor and received a giant peak voltage of 60 V, indicating the benefit of adding BNBT short-fiber into the PVDF devices. Besides, the enhanced output for the BNBT particles and fibers was briefly discussed. This work potentially gives a reference and opens a gate for the piezoelectric composite fibers in designing future sensors.


Host-microbiota interactions have increasingly been shown to play important roles in supporting host health . In humaLiu et al.). They found that microbial diversities were similar between these two techniques, suggesting SMS as an economical approach for future clinical tests. This study identified dominant species, such as Prevotella copri, Bacteroides dorei, and Bacteroides vulgatus, that might be responsible for the progression of colorectal diseases. These strains may serve as chassis to diagnose, or even engineered to treat colorectal diseases. Next-generation sequencing (NGS) technology was also demonstrated to be a powerful technique in the study by Chen and coauthors to detect fetal copy number variant for non-invasive prenatal testing . With increased read depth and reduced cost, it is foreseeable that NGS will have more contributions in the future.In order to modulate microbiota, we need to understand the composition of microbiota and their interactions with host. In this respect, Liu and coauthors used 16S rRNA gene amplicon and shallow metagenomic sequencing (SMS) to explore the gut microbiota directly from patient samples with colorectal disease . They discovered that abundance of membrane GLUT1 and cytoplasmic G6PD was greatly reduced and the GPR109a-AKT signaling pathway was vital in regulating these changes. More strikingly, they found that butyrate could promote chemotherapeutical efficacy of 5-fluorouracil on cancerous colonocytes, possibly due to exacerbated impairment of DNA synthesis. This study provides valuable information for the molecular mechanism of butyrate on glucose metabolism of colorectal cancer cells. These discoveries could promote the development of beneficial metabolites as therapeutical or adjuvant anti-cancer drugs. It is worth noting that butyrate could improve the antivirus capacity of human . They focused on LuxR, a bacterial quorum sensing regulator from Vibrio fischeri, which can be engineered to sense environmental signals and control overall microbiota behaviors. In this work, they identified a key residue, isoleucine-46, when modified to F or R, can have different binding affinity to the same substrate 3-oxo-hexanoyl-homoserine lactone. They implemented the designed transcription factors into genetic circuits in E. coli to detect target bacterial pathogens such as Yersinia pestis. The overall workflow showcased how synthetic biology, especially protein engineering and circuit design, can facilitate microbiota engineering for bio-therapeutical applications.Rather than relying on existing microbiota and molecular parts, with the advances in synthetic biology, we can design and engineer valuable regulatory parts as needed. Li and coauthors employed molecular dynamics simulation and the Molecular Mechanics Generalized Born Surface Area (MM-GB/SA) method to artificially designed transcription factors with different binding affinities . Chen and coauthors summarized impacts of the host-microbiome interactions on osteomyelitis pathogenesis, covering direct and indirect microbiome-associated osteomyelitis . Finally, Yang and coauthors reviewed the immune responses of severe COVID-19 in the gastrointestinal microenvironment and the gut-lung axis . This review highlighted the potential of using probiotics as an alternative drug to alleviate or even prevent severe COVID-19 outcomes.Several excellent reviews were also assembled for our readers in this topic. Zhang and coauthors provided a comprehensive review of the recent advances in understanding mechanisms of microbiomes as well as synthetic biology technologies that can be utilized to prevent, detect, and treat various diseases (We hope the articles and reviews collected in this research topic can show how fascinating this emerging field in the intersection of synthetic biology and microbiota is, and the great therapeutical potentials this field might offer. We want to take this chance to thank all the authors and reviewers for contributing to this special topic issue. As we are starting Volume II, we encourage and welcome more experts from this field to submit their research work to our topic.


The field of neuromuscular research has seen considerable recent advances in the molecular and cellular understanding of muscle biology, and the treatment of neuromuscular disease. These advances are at the forefront of modern molecular methodologies, often integrating across wet-lab cell and tissue models, dry-lab computational approaches, and clinical studies. The continuing development and application of multiomics methods offer particular challenges and opportunities, not least in the potential for personalized medicine. More than 500 different genes are known to be associated with neuromuscular disorders , and theThis Special Issue, entitled “Understanding Neuromuscular Health and Disease: Advances in Genetics, Omics, and Molecular Function”, encompasses some 15 publications from colleagues working on a diverse range of neuromuscular diseases, including Duchenne muscular dystrophy ,7,8,9, fThe search for genotype–phenotype correlations can be aimed at the improved understanding of disease ,5,7,9, bThe use of genomics and multi-omics approaches is a theme which itself cuts across the aims of current research, from the overlay of multiple omics data to achieve a global perspective and new understanding of Emery–Dreifuss muscular dystrophy , to the Aside from therapeutic strategy development, the use of biomarkers may be critical as disease trackers for the development of effective therapeutics , but alWe hope that studies such as these, that integrate modern molecular methodologies across cell and tissue models, computational approaches, and clinical studies, will continue to drive progress towards improved neuromuscular health and treatments for these often severe diseases.


Science, laying the groundwork for the field of molecular medicine. Over the past 70 years, chemistry has been one of the major forces pushing the field of molecular medicine to grow and evolve. Today, many important contributions made by chemists can be found in different subfields of molecular medicine, including molecular imaging, molecular diagnosis, molecular drug design and gene editing, et al. As in other areas of science and technology development, molecular medicine is thriving with active contributions of chemists and other molecular scientists.Molecular medicine aims to understand the molecular mechanisms underlying disease pathogenesis in order to develop suitable diagnostic strategies and disease interventions. From this perspective, chemists can and have to play a pivotal role in the field of molecular medicine as chemists are molecular scientists. In 1949, the internationally renowned chemist Linus Pauling published his seminal work entitled ‘Sickle cell anemia, a molecular disease’, in et al. summarize some recent progress in applying chemical biology to explore the molecular mechanisms of carcinogenesis, outlining several new strategies for chemistry to probe cellular activities. Ying-xuan Chen and Jing-Yuan Fang et al. discuss recent findings related to the crosstalk between microbiota and epigenetic modifications in colorectal cancer. Bing Su et al. review Sin1, a key adaptor molecule involved in the regulation and function of the mammalian target of rapamycin (mTOR) signaling pathway. Xinyuan Zhu and Deyue Yan et al. introduce recent advances in supramolecular designs of nanoscale drug delivery systems. In the perspective article by Xiawei Wei and Yuquan Wei et al., opportunities and challenges in the use of nanoparticles for nucleic acid therapeutics are discussed. Guangjun Nie and Yuliang Zhao et al. review some recent trends in nanomedicine design and further discuss challenges and opportunities in the development of next-generation nanomedicines. The research highlight article by Chaoyong Yang et al. reports a recent finding tracking the levels of circulating exosomal PD-L1 may help to predict the response of patients and identify the possible reasons for success or failure of anti-PD-1 therapy. An interview with Feng Shao and Weihong Tan is also included in this special topic. They share their personal views of the impact of chemistry on the biomedical sciences, in addition to their opinions on biomedical research methods and career development.This special topic features biomedical scientists, physicians and chemists, mostly from Shanghai Jiao Tong University, to showcase the influence of chemistry on the field of molecular medicine. In their review, Guo-Qiang Chen To effectively tackle challenging problems in molecular medicine, interdisciplinary approaches and collaborations are essential. We hope this special topic will inspire more researchers from different disciplines of science, engineering, pharmacy and medicine, as well as commercial companies, to join in the exciting field of molecular medicine.Finally, we would like to take this opportunity to express our gratitude to all the authors, reviewers, and the NSR editorial staff for their efforts in making this special topic possible.