From Operation Warp Speed to the lipid mRNA vaccine, the COVID-19 pandemic has been a watershed moment for technological development, production, and implementation. The scale and pace of innovation and global collaboration has likely not been experienced since World War II. This article highlights some of the engineering accomplishments that occurred during the pandemic. We provide a broad overview of the technological achievements in vaccine design, antibody engineering, drug repurposing, and rapid diagnostic testing. We also discuss what the future of these technologies and the future of large-scale collaborations might look like moving forward. It is here that we can draw the most meaningful comparisons to grasp the magnitude of growth that occurred during COVID-19.The urgent diagnostic, therapeutic, and protective equipment needs posed by the COVID-19 pandemic ushered in a wave of global collaboration that arguably has not been experienced since World War II. The contributions of scientists and engineers shaped the style of warfare and fundamentally changed the way that scientists, scientific institutions, and governments interacted.5 The difference was the mass production of penicillin. Alexander Fleming haphazardly discovered penicillin in 1925 as a mold growing in an overlooked petri dish.7 It would take more than a decade before those results were revisited because of the difficulty in purification. In 1939, a group of British chemists led by H. Florey were able to purify penicillin and published seminal papers demonstrating its efficacy in an animal model the following year.8 After this monumental publication, a flurry of clinical trials began. Doctors used purified penicillin to treat patients who had shown no efficacy with the existing treatment.8 Unfortunately, while there were some successes, many patients still succumbed to infection due to inefficient penicillin doses. Penicillin could be purified and concentrated, but it was still difficult to achieve sufficient amounts needed to treat a single patient, let alone an entire population. At this point, Florey began pursuing avenues to produce larger quantities of penicillin. This led him to collaborations with the US Department of Agriculture and pharmaceutical companies such as Merck, Pfizer, Abbot, and what is now Bristol Myers Squibb.6 The interactive collaboration led to fermentation research that increased penicillin yield, new batch reactors that facilitate high-volume production. Combined with the government financial backing from the US War Production Board,9 penicillin production went from enough to treat a single patient in 1942 to being able to deliver 2.5 million doses before the Battle of Normandy in 1944.10 Simultaneously, other countries began to increase their own production of penicillin. In this manner, the success of penicillin has direct connections with the speedy production and distribution of the mRNA vaccine.Many soldiers died from post wound infections such as gangrene. An estimated 15% of wounded soldiers died from infections in World War I compared to 3% in World War II.Although the COVID-19 pandemic was not a global political war, it was and remains a global biological challenge that demanded alliances and participation between academic and clinical medicine, industry, and government. This article will give an overview of the technological victors of the COVID-19 pandemic. The mRNA vaccine, SARS-CoV-2 antibody development, rapid testing diagnostics, and personal protective equipment (PPE) manufacturing were all critical to curbing the COVID-19 death toll. In addition, the article will highlight the materials research innovations that underpinned these developments.11 Of these, mRNA vaccines have received widespread attention both for being the fastest to market and the first mRNA-based vaccines approved for use in humans  addressed mRNA enzymatic degradation and low cell uptake, some of the biggest challenges in developing mRNA vaccines.12 The two mRNA vaccines currently approved for public use are mRNA-lipid nanoparticle (mRNA-LNP) vaccines that encode for the SARS-CoV-2 spike protein.13mRNA vaccine technology existed long before COVID-19 but remained a scientific prospect due to the formulation challenges and poor success in clinical trials.13 Since that drug’s launch in 2018, LNP delivery systems had fallen out of favor because of their side effects. The successful use of LNPs in SARS-CoV-2 vaccines has cast a renewed global spotlight on LNPs.Instead, research and funding had focused on other vaccine platforms that had proven safety and efficacy, including live attenuated viruses, recombinant proteins, or viral vectors. The COVID-19 pandemic led to significant urgency that pushed the innovation of mRNA vaccines forward, with lasting implications on our ability to fight future pandemics. Prior to mRNA-LNP vaccines, there was only one FDA-approved nucleic acid-LNP therapeutic, Alnylam’s Patisiran.14 Because this modification was already shown to work in other coronaviruses, scientists had an existing reference point when creating a stable spike protein for the SARS-CoV-2 vaccine. Prototype pathogens, in this case MERS-CoV and SARS-CoV, can be used to optimize vaccine designs for different viral families in anticipation of outbreaks.15 Previous research done with the prototype pathogen helped shorten the time span of vaccine development for SARS-CoV-2 by several months.The speed of mRNA vaccine development was first and foremost made possible by the previous research done with MERS-CoV and SARS-CoV. In developing vaccines for these viruses, stabilizing mutations were introduced to the mRNA, increasing the vaccine’s antibody recognition and immunogenicity.15 If the protein is an antigen, then sustained antigen availability can correlate with the intensity, duration, and cellular composition of the immune response.16 LNPs can also possess immunostimulatory properties, inducing immune responses comparable to other traditional vaccine adjuvants like Alum.12 The LNP platform is highly compatible with nucleic acid formulations, which made it readily available for incorporation with the SARS-CoV-2 mRNA sequence. In contrast, traditional vaccines require extensive manufacture of cell lines to produce attenuated viruses or clinical-grade protein subunits. As a result, mRNA-LNP vaccines are also more cost effective and scalable for the needs of a global pandemic. Further, mRNA-LNP vaccines are translated within the cell cytoplasm and as such has no risk of integrating into host chromosomes, unlike DNA-platform vaccines.17Using LNPs as the mRNA delivery platform also has additional benefits. Cellular uptake of mRNA-LNPs can vary by route of administration. In comparison to intravenous routes, mRNA-LNPS delivery via intramuscular and intradermal routes have exhibited higher protein expression.18 Other clinical studies have shown that the vaccine-elicited antibodies may be less effective at neutralizing variants with certain spike mutations. Pfizer-BioNTech’s as well as its competitors have begun to explore the significance of booster doses against SARS-CoV-2 variants, though as of the writing of this article, the data have not been published.19The “plug and play” nature of the mRNA-LNP vaccine platform has reduced the barrier to new or booster vaccine development timelines. Nonetheless, emerging SARS-CoV-2 variants have raised concerns about the effectiveness of previously developed and administered vaccines. These data are likely to change as the pandemic evolves; however, currently published clinical studies have demonstrated that mRNA-LNP vaccines elicit strong neutralizing antibody and CD4 + T cell responses are predicted predicted to help mount an effective immune response against SARS-CoV-2 as well as its variants.21 Antibodies treat infection via neutralization, opsonization, and complement activation. COVID-19 antibodies target the SARS-CoV-2 spike (S) protein because it is heavily involved in virus entry and infection of cells and transmission.22 Because mortality and complications seem to be associated with high viral loads, treatment with neutralizing antibodies may decrease the severity of the condition.23 Current methods include convalescent and monoclonal antibodies and, overall, studies suggest that this method of treatment is most effective for individuals with mild to moderate forms of the disease and are in the early stages of infection.25 Clinical trials with larger sample sizes are ongoing to continue examining their safety and efficacy in different groups with varying risk factors and stages of infection.Since vaccine-mediated adaptive immune responses take weeks to develop, individuals with high risks of contracting the virus may benefit from an immediate immunity obtained through passive antibody therapy.21 However, there is a lack of wide-scale randomized testing to assess the effectiveness of convalescent plasma therapy as a standard treatment for COVID-19, although trends in various clinical assessments suggest that they can be beneficial especially for those in early stages of infection.25 Challenges in convalescent plasma therapy include difficulties in collection and selection: plasma needs to be collected from donors and then analyzed for high IgG levels. Also, plasma may be contaminated with infectious agents and there are also risks associated with transfusion including circulatory system overload.20 However, analysis of convalescent plasma can be used to identify potential antibodies for monoclonal antibodies therapy development.20Convalescent antibodies are obtained from donors who recovered from infection with SARS-CoV-2. Convalescent antibodies have been used for treating infectious diseases when there is no specific treatment.23 Similarly, drugs Bamlanivimab and Etesevimab containing neutralizing antibody LY-CoV555 administered together helped to reduce mortality for those without hospitalizations.26 Casirivimab and Imdevimab are co-administered intravenously and can currently be used in patients with mild to moderate COVID-19 (starting from age 12 and more than 40 kg) who have risks of progressing into severe forms. Similar regulations for Bamlanivimab and Etesevimab exist;27 use of these drugs are prohibited for those with severe COVID-19 because observations suggest that complications may arise that cause the need for high oxygen flow or mechanical ventilation.The drugs Casirivimab and Imdevimab contain REGN-COV2 antibody cocktails that were effective in reducing viral load, especially in patients with high baseline viral load or those whose own adaptive immune response had not been initiated.28 Several strategies attempt to neutralize virus particles to reduce the risk of SARS-CoV-2 transmission. One such example is Engineering Water Nanostructures (EWNS), nanoparticle structs that contain reactive oxygen species that an deactivate airborne virus and/or microbes.30A less obvious winner in the COVID-19 therapeutics race is the advancement of aerosolized therapeutics, engineered particles that can engage with SARS-CoV-2 virus while in air circulation. Nebulizing monoclonal antibodies to facilitate lung delivery was growing in popularity, but probably accelerated during the COVID-19 pandemic.31 Nebulized, convalescent antibodies also had the benefit of dose sparing effects due to localized lung delivery.32 Aerosolized delivery of single-domain antibody fragments, or nanobodies, were also engineered.34 Nanobodies are advantageous since its smaller size reduces manufacturing costs.  PiN-21 is an example of a successful nanobody that reduced SARS-CoV-2 infection to levels below 0.1 ng/mL in in vitro models and decreased lung viral titers for 6-log reduction in in vivo hamster models.34 Synthetically designed nanobodies that bind SARS-CoV-2 spike protein, prevent binding with human ACE2 receptors.33 Nanobodies were rationally designed using techniques such as yeast surface-displayed synthetic libraries and affinity maturation,33 camelid immunization and proteomics modeling.35 The power of these high throughput synthetic library screening, integrated modeling, facilitated the fast and accurate development of new therapeutics to target emerging virus variants.36Inhalation was used as a therapeutic delivery route. The transition from intravenous to inhalation was commonly achieved by nebulization or the use of novel nanomaterial formulation strategies for optimal pulmonary delivery.37 Molecular testing involves detection of the viral genetic material and has largely used reverse-transcriptase real-time PCR (RT-PCR) for DNA amplification.38 While these tests have high accuracy, they also require specialized equipment and technicians and can also take hours for detection.39 Thus, rapid-antigen detection (RAD) tests  have been developed that do not require specialized equipment and can output results as fast as 30 min39  are running surveillance on assays available throughout the globe to ensure that they have high sensitivity and specificity (standard: ≥ 80% sensitivity and ≥ 97% specificity).41The accessibility of RAD tests has allowed for the development of diagnostic kits that are widely available to the public over-the-counter and through health care providers. In fact, the first self-testing kit was granted emergency use approval (EUA) by the FDA in November 2020 and since then the number of EUA testing kits have grown to 15 as of 3/26/2021 with many others across the globe with CE-IVD.44 The benefits to repurposing drugs are not new. These drugs are already FDA approved and have established manufacturing and distribution chains. Nonetheless, there are thousands of available therapeutics which have different molecular targets, varying purposes , as well as different drug types . To solve this challenge, computational tools have been implemented. Multiple studies were released that mapped the SARS-COV-2 protein interaction with human proteins to identify druggable proteins and existing compounds.45 Notably, one VINI in silico model found a combination of HIV drugs that could successfully inhibit SARS-CoV-2 spike glycoprotein.46The rapid need for therapeutics to treat moderate to severe COVID symptoms prompted the use of computational tools to speed up drug repurposing.47 The OpenData Portal is cited in numerous peer-reviewed papers and has successfully allowed researchers to identify SARS-CoV-2 potential drug candidates that were not but also allowed for the discovery of others that were previously unidentified.48 While these targets still have to be validated in vitro and through human clinical trials,44 this accelerated the decision-making process for drug discovery.Many of the computational models being employed are possible because of the open-source data sets of FDA approved drugs. While there are many, a great example is the NIH NCATS developed as an OpenData Portal, a compilation of SARS-CoV-2 screening related assays against all FDA approved drugs and related assay protocols.49 Even after the COVID-19 pandemic ends, mRNA vaccines will likely be a focus of vaccine development for prototype pathogens now that the mRNA-LNP platform is available and proven efficacious. It should be noted that the investment in technologies that had potential but whose application was not fully realized was critical preparation for the fast development of the COVID mRNA vaccines. The success of penicillin development during WW2 is just as valid of a lesson as the government support of mRNA vaccine development in 2020.The collaboration amongst science, industry, and government that occurred during COVID-19 was an enormous achievement. This presents a great opportunity to commemorate the successes and reflect on the advancements that will persist after the pandemic. The mRNA vaccine field has been reinvigorated and is now being applied to other infectious diseases.50 preceded the success of the lipid formulated mRNA vaccine. The use of mRNA as a vaccine system depended on decades-long research into the stabilization of mRNA nucleotides.51 Biocatalytic reactions research fueled protein engineering and the development of rationally designed antibodies and nanobodies.52 Moreover, the 3D manufacturing that drove the protective equipment pandemic needs was facilitated by basic research in polymer chemistry and stereolithography.53While it is easy to have a focus on the final product, the rapid enactment of COVID-19 related diagnostics and therapeutics was rooted in pre-pandemic basic research in biomaterials. Decades of lipid nanoparticle engineering54 The COVID-19 scientific and technological breakthroughs have led to unprecedented increases in budgets that will impact research funding even after the pandemic. In 2020, DOE and NIH budgets increased by $USD11.25 billion.55 NSF funding will potentially increase by $USD100 billion over the next five years, its largest increase since its founding in 1950.56 Similarly, the private sector has benefited. In 1944, the government funded success of penicillin catapulted Merck, Pfizer, Abbott, and what is now Bristol Myers Squib into a new pharmaceutical era.57 In 2020, Moderna went from an early-stage, high-risk biotech company worth $USD6.5 billion to having a market value of $USD100 billion.58Like the development and production of penicillin circa World War II, the speed and scale of the technological advancements achieved during the COVID-19 pandemic can be attributed to the funding of both technology and basic research. The US Department of Health and Human Services and the National Institutes of Health formed Operation WARP Speed to support the manufacturing and distribution of therapeutics, diagnostics, and protective equipment.59 In follow-up clinical trials, hydroxychloroquine had no therapeutic benefit,60 or in some studies, led to worse outcomes in COVID-19 patients.61 In addition, its use as a COVID-19 therapeutic created long-term supply shortages that affect other chronic patient populations such as individuals living with Lupus.62This is also a time to correct issues that may have been overlooked. The rush toward repurposing suitable COVID-19 therapeutics lead to the promotion of hydroxychloroquine. Interestingly, the hydroxychloroquine literature became highly contested following a flurry of retractions in high-tier publications. Several of these publications were based on electronic health records which were readily attainable but hard to verify.63 and decreased the years of potential life lost (YPLL) in non-Hispanic black and Hispanic individuals.64 In the United States, the distribution of COVID-19 vaccines brought a reprieve to the worst conditions of the still ongoing pandemic. However, this was not the case for many countries. Limited access to vaccines and other COVID medical interventions across countries have resulted in health care and economic inequalities.65 As countries raced to develop their own vaccines, there was a rise in vaccine nationalism.66 Moreover, with wealthy nations purchasing virtually all of the available vaccines, leaves people in less resourced places little to no opportunity to become vaccinated until at least 2022.66The speed at which funding resources were dispensed have left out underrepresented minority communities. COVID-19 exacerbated preexisting health disparities67 This has also been applied in the scientific realm. Convergence among nanomedicine, medical devices, and computational tools to develop pandemic preparedness pipelines.68 For better or worse, COVID-19 has shaped the next era of scientists, doctors, public health experts, and politicians. The rapid growth seen in the last two years will surely have impacts that last decades. It is the authors hope that science and society will be better for it.Regardless, the future is bright for multidisciplinary driven technology development. The COVID-19 pandemic has fostered a new era of convergence science, the integration of multiple disciplines and methods to solve complex problems. A broad example of this is the convergence of political, and public health systems to mount a multiscale preparedness plan for future pandemic threats.


Here, we review recent advancements in the development and characterization of PSD-95-targeted CPPs and propose the combination of these two approaches to improve treatment of stroke and other excitotoxicity-associated disorders.Therapies for stroke have remained elusive in the past despite the great relevance of this pathology. However, recent results have provided strong evidence that postsynaptic density protein-95 (PSD-95) can be exploited as an efficient target for stroke neuroprotection by strategies able to counteract excitotoxicity, a major mechanism of neuronal death after ischemic stroke. This scaffold protein is key to the maintenance of a complex framework of protein interactions established at the postsynaptic density (PSD) of excitatory neurons, relevant to neuronal function and survival. Using cell penetrating peptides (CPPs) as therapeutic tools, two different approaches have been devised and advanced to different levels of clinical development. First, nerinetide (Phase 3) and AVLX-144 (Phase 1) were designed to interfere with the coupling of the ternary complex formed by PSD-95 with GluN2B subunits of the N-methyl-D-aspartate type of glutamate receptors (NMDARs) and neuronal nitric oxide synthase (nNOS). These peptides reduced neurotoxicity derived from NMDAR overactivation, decreased infarct volume and improved neurobehavioral results in different models of ischemic stroke. However, an important caveat to this approach was PSD-95 processing by calpain, a pathological mechanism specifically induced by excitotoxicity that results in a profound alteration of survival signaling. Thus, a third peptide (TP95 Stroke is the second leading cause of death in the world. In 2019, there were 12.2 million incident cases, 101 million prevalent cases and 6.55 million deaths from stroke  . T. T2+-infThe central role played by PSD-95 in neuronal survival and death makes this protein a very interesting target for the development of neuroprotective strategies for stroke and other pathologies associated to excitotoxicity. Two different approaches targeting PSD-95 are currently under investigation. A very relevant strategy is directed toward preventing the induction of PSD-95-dependent pro-death signaling and neurotoxicity by dissociation of GluN2B–PSD-95–nNOS complexes. However, an important caveat to this approach is PSD-95 calpain-processing, a pathological mechanism that results in a profound alteration of survival signaling. Thus, the stabilization of PSD-95 is being explored as an alternative, and possibly complementary, neuroprotective strategy. Here, we review three very promising cell-penetrating peptides (CPPs) exploiting these neuroprotective strategies that have advanced to different levels of development.Treatment of CNS pathologies with therapeutic molecules is intrinsically difficult due to the presence of the blood–brain barrier (BBB). An innovative idea at the center of pharmaceutical research is shuttle-mediated delivery of the therapeutic molecules by conjugation to compounds able to cross the BBB, among them, small naturally derived peptides. CPPs are defined as a group of relatively short peptides (4–40 aa) of different chemical characteristics (frequently positive charged) that can get through the BBB and cellular membranes by means of different mechanisms, mainly endocytosis . MoreoveThe development of nerinetide by Dr. Tymianski’s group has set the standard for how to navigate from basic research to clinical translation in human stroke. Nerinetide, previously known as NA-1 or Tat-NR2B9c, is an eicosapeptide formed by the Tat sequence followed by the 9 C-terminal residues of NMDAR-GluN2B subunit, containing the PDZ-ligand A. ConsidA Phase 2 clinical trial assay called ENACT  was completed in 2012. This study was aimed at evaluating the safety and efficacy of nerinetide for reducing embolic stroke in the context of an endovascular treatment of intracranial aneurysms . A doublNevertheless, the drug–drug interaction between alteplase and nerinetide is still problematic for stroke patients who qualify for alteplase treatment and justifies further investigation. The aminoacidic sequence of nerinetide is susceptible to cleavage by plasmin, a serine protease generated from circulating plasminogen by alteplase . Thus, nThe dissociation of GluN2B–PSD-95–nNOS complexes, without affecting the physiological functions of the NMDARs, has drawn the interest of other research groups that have developed different compounds having mechanisms of action similar to those of nerinetide. Some of them are small molecule inhibitors targeting the PDZ domains of PSD-95, such as IC87201 or ZL006, recently reviewed elsewhere . The groUsing a mouse model of permanent MCAO, they demonstrated that treatment with an intravenous dose of AVLX-144 (3 nmol/g) 30 min after ischemia protected against brain damage and improved neuromuscular function. Interestingly, nerinetide did not show significant effects in this mice model when used at this concentration , but it The potential of AVLX-144 as neuroprotectant has encouraged further studies towards clinical translation. A Phase 1 study in healthy volunteers to evaluate safety, tolerability and pharmacokinetics is currently underway . This is a randomized, double-blind, placebo-controlled, single ascending dose study that plans to first analyze AVLX-144 safety and tolerability in healthy male subjects (age 20–50 years old). Once establishing the drug dose, healthy elderly subjects  will be analyzed.457) containing TrkB-FL sequences controlling calpain-processing of this neurotrophin receptor was developed and shown to sustain survival signaling in excitotoxic conditions. This neuroprotective peptide decreased the infarct size and improved the neurological outcome in a mice model of permanent stroke [29, TP95259 and TP95414) containing the PSD-95 calpain-cleavage sequences that potentially could specifically interfere with this processing. This strategy, aimed at preventing the pathological processing of this specific calpain substrate, is not expected to interfere with the physiological functions of PSD-95. The use of generic calpain inhibitors as an alternative strategy was ruled out because they might inhibit the physiological actions of this protease and produce unwanted side effects [The second approach to stroke treatment targeted to PSD-95 is the prevention of the characteristic processing of this protein induced by NMDAR overactivation. A previous work demonstrated that it is possible to design CPPs that interfere with the processing of a specific calpain-substrate in excitotoxicity and produce stroke neuroprotection . Thus, at stroke . Therefot stroke . This in effects .414, containing PSD-95 residues 414–427  10 min after injury tends to interfere with PSD-95 processing and decrease infarct volume. Despite this modest decrease in infarct volume (15%), TP95414 significantly improves the neurological function of treated animals. It is interesting that no effects were observed for a peptide concentration of 10 nmol/g, a result supporting the importance of dose–response experiments and suggesting that levels of PSD-95 have to be maintained in a correct range to support neuronal survival after brain damage.Further studies have demonstrated that peptide TP95penumbra  defies tAltogether, these results unveil PSD-95 stabilization as an attractive and highly relevant target for stroke therapy and perhaps for other important neurological conditions different from stroke, likewise associated with excitotoxicity.The development of the neuroprotective peptides reviewed here has been guided by a compromise with rationally designed molecules that are able to challenge specific pathological mechanisms activated in neurological diseases, stroke included. We have learned very important lessons from the past use of antagonists of glutamate receptors for stroke treatment. They lacked specificity, and thus, their administration affected both physiological and pathological receptor functions, producing unwanted side effects. Therefore, efforts in the field of neuroprotection have focused on the development of brain accessible molecules able to specifically target proteins or mechanisms downstream of NMDAR overactivation related to the pathological state. A thorough delineation of the dual roles of NMDARs in neurons has identified critical downstream death-signaling pathways that can be specifically inhibited without blocking the NMDARs ,25. Thes2+-influx, these peptides reduce neuronal surface expression of NMDAR subunits and the activity and/or surface expression of other ion channels and receptors. It has been also suggested that they target mitochondria, stabilize proteins, inhibit proteolytic enzymes, induce pro-survival signaling, scavenge toxic molecules and reduce oxidative stress [Many research groups have chosen CPPs to deliver specific therapeutic molecules to the CNS, and some of these neuroprotective peptides, as mentioned before, have reached clinical trials. Several other specifically targeted peptides are currently being tested in preclinical models of disease and have great translational potential. In a different approach, cationic arginine-rich peptides have been proposed as neuroprotective agents for stroke and other excitotoxicity-associated disorders. Thus, it has been recently shown that poly-arginine peptide-18 (R18) induces a decrease in brain injury and modestly improves functional outcome in a nonhuman primate stroke model . It has e stress . Such a 414 treatment, PSD-95 levels can be maintained in the damaged brain, but the formation of the trimeric complex and NO production would not be inhibited, thus limiting the therapeutic effect of this neuroprotective peptide. Therefore, we propose that the combination of both strategies could result in a more powerful neuroprotective effect since promotion of PSD-dependent pro-survival pathways and inhibition of pro-death actions would take place at the same time. This combined neuroprotective approach could be highly relevant for stroke therapy.We have summarized here results unveiling two different approaches for stroke neuroprotection directed at PSD-95: uncoupling of the NMDAR–PSD-95–nNOS complex and prevention of PSD-95 downregulation. Treatment with nerinetide or AVLX-144 alone will inhibit pro-death pathways activated by coupling of the NMDARs and nNOS in stroke but would not affect PSD-95 processing by calpain, still leading to profound changes in PSD and synapses B. AdditiIn this article, we reviewed evidence demonstrating that PSD-95 is a promising target for ischemic stroke therapy, as well as for other CNS disorders. The usage of CPPs able to block PSD-95 pro-death functions and/or favor the maintenance of this protein’s pro-survival activities during excitotoxicity have a great potential for neuroprotection.


PLOS Medicine addresses an evolving, poorly studied, and important area of health policy and planning related to the care of patients who survive hospitalization for COVID-19 [Emerging evidence suggests that approximately 10% of people who survive Coronavirus Disease 2019 (COVID-19) will have lingering symptoms that negatively affect their quality of life, ability to work, and function . HoweverCOVID-19 .At face value, the scope of the COVID-19 pandemic is enormous. Within 2 years, nearly 300 million people have been infected with the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus, and more than 5 million people have died from it . More seBhaskaran and colleagues studied over 164,000 hospitalized adults with COVID-19 matched to an “active control” group of adults hospitalized with influenza and to general population controls. They compared the medium- and long-term risks of hospital admission and death across the 3 study groups. The main findings were that people discharged following hospitalization for COVID-19 had a 2-fold higher associated risk for rehospitalization and death than the general population and similar risks compared to those hospitalized for influenza. These outcomes were most pronounced in the first 30 days following discharge yet remained substantially elevated over time. Further, those hospitalized with COVID-19 were more likely to be rehospitalized or die from mental health or cognitive-related causes, especially if they had preexisting dementia, compared to those hospitalized with influenza.Initial hospitalization with COVID-19 represents a crucial touch point within the healthcare system. The study by Bhaskaran and colleagues sheds important light on the health service needs of patients who survive hospitalization for COVID-19. It further helps disentangle the effects of hospitalization from respiratory viral infection on important outcomes. The current work builds on similar findings from a recent study of 47,780 hospitalized adults with COVID-19 who survived to discharge with a mean follow-up of 140 days .There were also noteworthy limitations of Bhaskaran and colleagues’ study. First, cause-specific outcomes among adults with COVID-19 may be artificially higher than those with influenza due to availability bias. Put simply, patients and providers may be much more aware of COVID-19 and its complications, including those related to return to hospital, than might be the case for those with pneumonia or even confirmed influenza. Second, the study used administrative data from primary care. While 98% of the population in England are registered with a general practice , there are some geographical differences in the use of the OpenSAFELY platform, which may introduce the potential for selection bias. Third, this study was conducted in a high-income nation with substantial resources to support patients following infection with COVID-19. The generalizability of these findings to middle- and low-income nations, or those with limited resources, is unknown.The study by Bhaskaran and colleagues has clear applications to healthcare resource planning and policy in the care of individuals who survive COVID-19. This suggests a substantial added burden on global healthcare systems. It further builds on our evolving knowledge of the post-COVID-19 condition and its lingering impacts, including on many previously healthy adults in their prime years of productivity. Still, a wealth of research is required to develop prediction tools to proactively identify and support the healthcare needs of survivors, including end-of-life care, develop new strategies to prevent and treat the post-COVID-19 condition, and encourage interprofessional teams to provide longitudinal care through innovative health policy interventions.Early pandemic public messaging strategies focused on flattening the peak of the acute COVID-19 infection curve to preserve healthcare system capacity and its ability to deliver high-quality care. These efforts were generally successful. To preserve ongoing system capacity and provide high-quality patient care, the long COVID tail does not require further flattening, but rather demands new clinical and health policy strategies to address its potential for long-term suffering. Here, we must recognize that the head of the pandemic often demands our immediate attention, but we must not ignore its long and deadly tail.


Bridge displacement measurements are important data for assessing the condition of a bridge. Measuring bridge displacement under moving vehicle loads is helpful for rating the load-carrying capacity and evaluating the structural health of a bridge. Displacements are conventionally measured using a linear variable differential transformer (LVDT), which needs stable reference points and thus prohibits the use of this method for measuring displacements for bridges crossing sea channels, large rivers, and highways. This paper proposes a reference-free indirect bridge displacement sensing system using a multichannel sensor board strain and accelerometer with a commercial wireless sensor platform (Xnode). The indirect displacement estimation method is then optimized for measuring the structural displacement. The performance of the developed system was experimentally evaluated on concrete- and steelbox girder bridges. In comparison with the reference LVDT data, the maximum displacement error for the proposed method was 2.17%. The proposed method was successfully applied to the displacement monitoring of a tall bridge (height = 20 m), which was very difficult to monitor using existing systems. Structural health monitoring (SHM) provides information about the current condition of a structure, thereby assisting decision-making for operation and maintenance. Continuous SHM allows early-stage damage detection, reduces the downtime, and prevents potential failure during operation. Vertical displacement induced due to traffic loading is regarded as a crucial measurement in bridge health monitoring because the displacement change is negligible under ambient traffic loads. Ambient displacement monitoring detects any structural damages or stiffness degradation of a bridge. Traditionally, the displacement is measured using a linear variable differential transformer (LVDT), a contact-type sensor that provides accurate displacements. However, the installation of LVDTs to bridges is limited, as they must be affixed on a stationary reference ,2,3. TheTo address the limitations of reference-based methods requiring stationary reference for sensor installation, wireless smart sensors have been developed to measure the structural responses and to estimate displacement indirectly. Acceleration-based displacement estimation for reference-free measurement can be performed through direct integration in the frequency domain using finite impulse response (FIR) filters. Although FIR filters can eliminate the error from double integration of acceleration, only dynamic displacement can be reconstructed ,14,15.In addition to acceleration, strain measurements can be used to estimate reference-free displacement based on strain-displacement relationships ,17,18. STo overcome the issues of strain and acceleration, data-fusion-based, reference-free indirect displacement estimation was developed to estimate bridge displacements . ReferenThis study develops a practical indirect displacement sensing system with multimetric sensing that simultaneously measures three-channel accelerations and three-channel strains. First, the multimetric board capable of simultaneously observing the three-axis acceleration and three-channel strains was proposed. Second, to enhance the accuracy of the displacement estimation, the placements of the three-channel strains were optimized in a numerical study. The indirect bridge displacement estimation method was optimized for three distributed strain measurements and one acceleration measurement. The proposed system was realized using a commercial wireless sensor platform called Xnode ,25,26, aThe remainder of this paper is organized as follows. A multimetric sensing of acceleration and strain is required for multiple and high-sensitivity vibration and strain sensing. The accelerometer should be carefully designed to capture the vibrations not only of lightly damped steel structures but also of highly damped concrete structures. Multiple small strain responses (<1 us) must be captured on the bridge surface. The wireless sensor platform on which the developed multimetric sensor board is integrated should be carefully selected. Therefore, when developing a multimetric sensor board for wireless sensors, the following three typical design challenges must be considered: (1) identifying an appropriate wireless sensor platform with high processing capability and low power consumption; (2) designing a board with a high-resolution accelerometer and strain sensor with power management; and (3) designing a stable and low-noise printed circuit board (PCB) for physically integrating the accelerometer with the wireless sensor platform.To measure multiple accelerations and strains at high sampling rates , the base wireless sensor platform requires sufficient processing capability for implementing real-time digital filtering and recording. For the wireless sensor platform for multimetric sensing, we selected Xnode for its high processing speed and sufficient synchronous dynamic random access memory (SDRAM) size for real-time data processing. The standard Xnode consists of three main boards: the processor, radio/power, and sensor boards a. The prThe radio/processor board has a 2.4-GHz Zigbee radio for low-power wireless communication (Atmel AT86RF233) . After aThe configuration of the developed multimetric board is shown in A three-channel-strain sensing circuit was designed using a quarter-bridge, which is a type of Wheatstone bridge . VEXT isε is the strain. The resistance change in the strain gauge is linearly related to the voltage difference. Using Equation (1) with GF = 2, the voltage corresponding to a strain change of 1 The voltage difference between points A and B was calculated asBased on the relationship between the GF and the resistance change given by Equation (3), we can compute the corresponding strain change:Comparing the actual voltage difference before and after connecting the known shunt resistance, we obtain the conversion factor between the voltage and strain units.Note that the half-bridge can be configured by replacement resistance between C and D in To handle the limited power resources available on the wireless sensor, a power management circuit was developed to control the power on demand. The most power-consuming part is strain sensing, where one 120-Ω strain gauge draws The noise level of the developed multimetric board was validated via a laboratory-scale evaluation. This test confirmed whether the PCB met the specified accelerometer and strain performances. The resistance and GF of the strain gauge were 120 Ω and 2.1, respectively. A strain gauge was attached at the top of a 1.5-high cantilever beam. The integrated multimetric sensor board with Xnode was deployed at the support of the beam and measured at a sampling rate of 100 Hz. The conversion factor obtained in the shunt calibration was 48,287 This section estimates the performance of the reference-free indirect displacement from the strain and acceleration measurements, and investigates the optimal sensor location for the three strain measurements available on the multimetric sensor board through numerical analysis.The displacement can be indirectly estimated from the vertical acceleration and three-channel strains measured using the developed multimetric board. Park et al.  proposedi.x, respectively. Here, A numerical study was conducted on the locations of the three strain measurements available for the developed multimetric sensor board. The sensor deployment was symmetric about the center, as suggested by Park et al. . SpecifiSimulations were performed on simply supported beams of various lengths. The cross section of the model was 2 m × 1 m (width × height), the modulus of elasticity was 210 GPa, the density was 7850 n-element Bernoulli beam, where n equals the length of the beam. For instance, a 30–m long beam comprised 30 elements and 29 nodes for simulating the acceleration, strain, and displacement. The estimated displacement in the proposed method was derived by simulating the vertical acceleration of the beam center and the three strain channels. The estimated displacement was then compared with the exact displacement obtained in the simulation. Three cases with different beam lengths  were simulated using symmetric strain-gauge deployments. One gauge was located at the center of the bridge while the other gauges were deployed equidistant from the center. In the simulation, the distance between the center and the two side gauges was manipulated, and the estimated displacement was compared with the exact displacement. For instance, along the 30-m long beam, the off-center gauges were located at 1–14 m from the center. At each strain measurement location, the acceleration and strains were simulated 100 times under various moving loads and the displacement was estimated. The error was defined as the root mean square error (RMSE) of the difference between the estimated and exact displacement divided by the maximum displacement (Equation (6)). The errors in the 100 simulations were averaged for comparison.The simply supported beam was modeled using an L from the center, where L is the bridge length. The optimal location of the strain measurements  was 0.2–0.25 × L from the center. For example, on the 30-m long bridge, the optimal position was 0.2 × 30 m  from the center. Interestingly, the measurements of the location of the strain from the center did not critically affect the error (the average error increment was 1–2%). At the sites where sensor installation was difficult, the strain sensors were located close to the center for the estimation of displacement.The proposed reference-free indirect displacement sensing system was experimentally validated on a prestressed concrete bridge located at Gwangju-si, South Korea. The experimental setup is presented in A second experiment was performed on a 40-m long steel-box girder bridge located at Gapyeong-gun, South Korea. An overview of the experimental setup is presented in To demonstrate the efficiency of the proposed cloud-based monitoring system, the developed sensor was instrumented on a tall (20-m high) concrete bridge located at Yeongdong-gun, South Korea. As the proposed indirect displacement sensing system is applicable to any type of bridge, the Geumkok Bridge was selected as the testbed because, to the best of our knowledge, its displacement has not been previously monitored. The developed sensor was installed beneath the bridge deck, where it could detect ambient vibrations. The sensor and strain gauges were installed and instrumented by a bridge inspection truck . The spaThe scaling factor was obtained from the strain and acceleration measurements using Equation (5). The structural responses with unknown input were monitored via video recordings during the measurements. In this paper, we developed and evaluated a reference-free indirect displacement sensing system. The system integrates a multimetric board that measures three strains and the three-axis acceleration using a commercial wireless smart sensor. The indirect displacement estimation method was optimized for three strain measurements and one acceleration measurement. In actual bridge monitoring, the number of wireless signals are inevitably reduced and the communication among multiple wireless sensors becomes difficult. For this reason, the proposed sensing system uses a single sensor unit for reference-free bridge displacement estimation.The developed multimetric board simultaneously observed the three-axis acceleration and three-channel strains and estimated the reference-free displacement.To enhance the accuracy of the displacement estimation, the placements of the three-channel strains were optimized in a numerical study.The main contributions of this study are as follows:When developing the multimetric sensor board for wireless sensors, the following design challenges were addressed: (1) the board required a high-resolution accelerometer and strain sensor with power management; (2) the board was integrated with three Wheatstone bridges for three-channel strain sensing; and (3) an automated shunt calibration was developed and implemented in the software. Furthermore, the optimal location of strain sensing for the estimation of indirect displacement (0.2–0.25 of the span length) was numerically validated. For validation purposes, the developed multimetric board was integrated with the commercial wireless sensor platform Xnode. The integrated system was experimentally validated on two differently constructed railroad bridges and the estimated displacements were compared with those of LVDTs. On the Keukrak and Jojungchun Bridges, the maximum displacement errors were 2.17% and 0.17%, respectively. Finally, the developed system was applied to a tall (20-m high) bridgewhich was very difficult to monitor by using existing systems. The indirect displacements were successfully obtained, confirming that the proposed system can capture very small (<0.1 mm) reference-free displacements.Future work is underway to develop ambient bridge displacement monitoring by expanding the proposed sensing system to cloud-based bridge management, where sensor data is uploaded in a cloud database and data processing for displacement estimation can be implemented in an autonomous manner.


Ureaplasma spp. with a lower burden than those with intra-amniotic infection (p = 0.03). The intensity of intra-amniotic inflammatory response measured by interleukin-6 was higher in women with colonization than in those with negative amniotic fluid . Women with colonization had higher rates of acute inflammatory placental lesions than those with negative amniotic fluid. In PPROM, colonization, caused mainly by microorganisms from the lower genital tract, might represent an early stage of microbial invasion of the amniotic cavity with a weak intra-amniotic inflammatory response.To determine the main clinical characteristics of preterm prelabor rupture of membranes (PPROM) complicated by colonization of the amniotic cavity . A total of 302 women with PPROM were included. Transabdominal amniocentesis was performed and amniotic fluid was assessed. Based of microbial invasion of the amniotic cavity and intra-amniotic inflammation (interleukin-6 ≥ 3000 pg/mL), the women were divided into following groups: intra-amniotic infection, sterile intra-amniotic inflammation, colonization of the amniotic cavity, and negative amniotic fluid. Colonization was found in 11% (32/302) of the women. The most common bacteria identified in the amniotic fluid were  PPROM is characterized by a breach in the barrier between the intra-amniotic and vaginal/cervical environments4. Therefore, PPROM can become complicated by the ascension of microorganisms from the vagina/cervix, thus leading to microorganisms in the amniotic cavity, termed microbial invasion of the amniotic cavity9. Microorganisms in the amniotic fluid are recognized by specialized pattern recognition receptors localized on the amniotic epithelium or in the amniotic fluid13. The activation of these receptors triggers a well-orchestrated intra-amniotic inflammatory response leading to the elevation of the concentrations of various inflammatory mediators (intra-amniotic inflammation), along with the attraction of immune cells into the amniotic fluid, with the ultimate goal of solving the microbial threat16.Preterm prelabor rupture of the membranes (PPROM) is defined as a rupture of the fetal membranes, with leakage of amniotic fluid before the onset of regular uterine activity earlier than 37 weeks of gestation16, with preterm labor with intact membranes17, and those with a short cervix18. This condition is typically considered as either: (1) contamination of the amniotic fluid with skin bacteria during amniotic fluid sampling and/or during pre-analytical processing of amniotic fluid samples or (2) colonization of the amniotic cavity (colonization) with microorganisms from the lower genital tract in the absence or presence of a weak intra-amniotic inflammatory response20.Aside from the "classical" scenario, called intra-amniotic infection, in which the presence of microorganisms in amniotic fluid is associated with intra-amniotic inflammation, an alternative situation, where the presence of microorganisms in the amniotic fluid is not related to intra-amniotic inflammation, has been previously reported among women with PPROM21 and (2) microbial invasion of the amniotic cavity seems to be a consequence of PPROM22.The cause of microbial invasion of the amniotic cavity without intra-amniotic inflammation in PPROM is still under debate; however, colonization is prioritized because (1) the breach in the barrier between the lower genital tract and the amniotic cavity might enhance the ascension of vaginal/cervical bacteria into the amniotic fluid21, colonization is yet to be fully characterized. Therefore, it is essential to fill this knowledge gap and thoroughly investigate the clinical characteristics and significance of colonization to help clinicians deal with PPROM complications.In contrast with intra-amniotic infection and sterile intra-amniotic inflammation in PPROM pregnanciesTherefore, the main aims of this study were (1) to characterize the microbial composition of amniotic fluid from PPROM with colonization, (2) to evaluate the main clinical characteristics of colonization, and (3) to describe the short-term morbidity of newborns from women with colonization.Overall, 302 women with singleton pregnancies with PPROM were included in this study. The clinical definitions are shown in Table p = 0.27). The most common microorganism identified in the amniotic fluid from women with colonization and intra-amniotic infection was Ureaplasma spp. . Women with colonization had a lower load of Ureaplasma spp. DNA in the amniotic fluid than women with intra-amniotic infection  and those with sterile intra-amniotic inflammation .Gestational age at rupture of membranes differed among the subgroups of the women (p = 0.76). However, women with colonization had a shorter latency interval than women with sterile intra-amniotic inflammation . Comparisons with women with intra-amniotic infection were not made due to a different management approach (active management beyond the 28th week of gestation).Latency interval (hours) from rupture of membranes to delivery in women with preterm prelabor rupture of membranes with respect to intra-amniotic infection, sterile intra-amniotic inflammation, colonization of the amniotic cavity, and negative amniotic fluid is shown in Fig. p < 0.0001; Fig. p = 0.04; Fig. Concentrations of interleukin (IL)-6 in amniotic fluid differed among the subgroups of the women . No differences in maternal inflammatory response were found in the comparisons between women with sterile intra-amniotic inflammation  and negative amniotic fluid .Women with colonization had a lower intensity of maternal inflammatory response measured by C-reactive protein (CRP) and white blood cell (WBC) count than women with intra-amniotic infection  of the women (five missing findings were from women with negative amniotic fluid). The rates of acute histological chorioamnionitis (HCA), funisitis and inflammation of the amnion differed among the subgroups of the women  the gestational age at rupture of membranes in women with colonization was similar to that of women with negative amniotic fluid but higher than in those with intra-amniotic inflammation; (4) the intensity of intra-amniotic inflammatory response (IL-6 concentrations) was higher in women with colonization than in those with negative amniotic fluid; (5) the rates of acute inflammatory placental lesions was higher in women with colonization than in those with negative amniotic fluid; and (6) short-term neonatal morbidity did not differ between newborns from PPROM with colonization and those with negative amniotic fluid.The principal findings of this study are as follows: (1) colonization was found in 11% of PPROM pregnancies; (2) 20. The rates of these phenotypes might differ among cohorts of women with PPROM due to differences in distribution of gestational age at rupture of membranes, race/ethnicity, and latency interval between ruptured membranes and sampling. In this study, the rates of intra-amniotic infection and colonization were almost equal . The rate of colonization found in this study was similar to that reported previously (12%) in the study by Romero et al.16; however, the rate of intra-amniotic infection was lower than that reported previously (12% vs. 29%)16. These results show that colonization can be responsible for a significant amount (30–50%) of cases with microbial invasion of the amniotic cavity in PPROM pregnancies. This observation is of clinical significance. However, we must be aware that cases with colonization remain clinically hidden unless the assessment of both microbial invasion of the amniotic cavity and intra-amniotic inflammation is employed. This is in direct contrast to cases with intra-amniotic infection , for which a less expensive and less time-consuming assessment of intra-amniotic inflammation is sufficient.Microbial invasion of the amniotic cavity represents a condition with two clinical phenotypes: (1) intra-amniotic infection when intra-amniotic inflammation is present and (2) colonization/contamination when intra-amniotic inflammation is absentUreaplasma spp. are the most common pathogens in the amniotic fluid from PPROM23. These low virulent commensal bacteria, commonly found in the vagina and/or cervix are among the smallest self-replicating microorganisms that can grow independently26. In this study, the rate of Ureaplasma spp. in women with colonization reached 60%. In addition, a vast majority of microorganisms, other than Ureaplasma spp., identified in the amniotic fluid from this subset of women, were bacteria commonly present in the vagina, cervix, or rectal niche. These observations support that ascension of the bacteria from the lower genital tract might cause colonization in women with PPROM.There is solid evidence that Ureaplasma spp. varies from hundreds to billions of copies of DNA per milliliter of amniotic fluid in PPROM and it is higher in women with intra-amniotic infection than in those with colonization21. Accordingly, a lower burden of Ureaplasma spp. was observed in women with colonization than in those with intra-amniotic infection in this study. Given the dose-dependent relationship between intra-amniotic inflammatory response (measured by IL-6 concentrations) and the amniotic fluid burden of Ureaplasma spp. in PPROM pregnancies28, it is likely that women with colonization, caused by Ureaplasma spp., could not elicit an intra-amniotic inflammatory response intense enough to pass a clinical threshold for intra-amniotic inflammation. In addition, clinicians should be aware of the following facts: (1) intra-amniotic inflammation is expressed as a categorical condition (present/absent) and (2) IL-6 is a physiological constituent of amniotic fluid  compared to those with intra-amniotic infection, sterile intra-amniotic inflammation, and negative amniotic fluid, in which the ranges were almost two-fold higher. These findings might extend our previous observation, where an intensive intra-amniotic inflammatory response to amniotic fluid bacteria, characterized by concentrations of multiple inflammatory-related proteins in amniotic fluid, was not found beyond gestational 32 weeks29.There is evidence that the rate of intra-amniotic infection in PPROM pregnancies decreases with advanced gestational age31. This condition is typically followed by the attraction of neutrophils towards the amniotic cavity from the intervillous space of the placenta into the chorionic plate and/or from the decidua into fetal membranes, leading to the development of acute inflammatory lesions in the placenta32. In this study, both intra-amniotic inflammatory responses, measured by concentrations of IL-6 in the amniotic fluid and the presence of acute inflammatory placental lesions, were assessed. Collectively, women with colonization had a higher intensity of intra-amniotic inflammatory response and rates of acute inflammatory placental lesions than women with negative amniotic fluid. This observation further supports the hypothesis mentioned above that colonization in PPROM represents an early stage of microbial invasion of the amniotic cavity with a weak intra-amniotic inflammatory response that is not intense enough to pass a clinical threshold for intra-amniotic inflammation.The intra-amniotic inflammatory response is associated with elevated concentrations of various cytokines, chemokines, and other inflammation-related proteins and lipids in the amniotic fluid33. To extend this knowledge, the selected aspects of short-term neonatal morbidity were investigated in this study. After adjusting for gestational age at delivery, no differences were found between newborns from PPROM with colonization and negative amniotic fluid.It remains debatable whether the microbial invasion of the amniotic cavity and/or intra-amniotic inflammation, after correction for gestational age at delivery, is associated with worse neonatal outcomesUreaplasma spp., M. hominis, and Ch. Trachomatis; non-specific PCR for 16S rRNA; and aerobic/anaerobic cultivation were employed in this study. Finally, short-term neonatal outcomes were available for all newborns in the PPROM cohort.This study had several strengths. First, a relatively large cohort of women with singleton pregnancies complicated by a well-defined clinical phenotype of spontaneous preterm labor (PPROM) with available information about the intra-amniotic environment and histopathology of the placenta was used. Second, a thorough assessment of microbial invasion of the amniotic cavity consisting of specific PCR for Ureaplasma spp. DNA, but not all bacterial DNA, in the amniotic fluid were evaluated in this study. This shortcoming prevented us from assessing whether the burdens of amniotic fluid bacteria other than Ureaplasma spp. differ between women with colonization and those with intra-amniotic inflammation. However, in a recent study on women with preterm labor with intact membranes, microbial and fungal burdens in the amniotic fluid were lower in those with microbial invasion of the amniotic cavity without intra-amniotic inflammation than in those with intra-amniotic inflammation17. Last, the long-term outcomes of infants from PPROM pregnancies were not available.This study also has limitations that are worth mentioning. First, this study consisted of a homogeneous population of Caucasian women living in the eastern part of the Czech Republic. This prevents the findings of this study from being generalized to populations with racial/ethnic disparities. Second, only loads of In conclusion, colonization in PPROM, caused mainly by microorganisms from the lower genital tract, might represent an early stage of microbial invasion of the amniotic cavity with a weak intra-amniotic inflammatory response that is not intense enough to pass a clinical threshold for intra-amniotic inflammation.This retrospective study included all pregnant women admitted to the Department of Obstetrics and Gynecology of the University Hospital Hradec Kralove, Czech Republic, between December 2018 and July 2021, who met the following criteria: (1) age ≥ 18 years, (2) PPROM between gestational ages 24 + 0 and 36 + 6 weeks, (3) singleton pregnancy, and (4) transabdominal amniocentesis. The exclusion criteria were as follows: (1) pregnancy-related and other medical complications , (2) congenital or chromosomal fetal abnormalities, (3) signs of fetal hypoxia, and (4) significant vaginal bleeding.Gestational age was determined based on a first-trimester ultrasound scan. PPROM was diagnosed based on visual confirmation of amniotic fluid pooling in the posterior vaginal fornix using a sterile speculum for examination, or by determination of the presence of insulin-like growth factor-binding protein in the vaginal fluid  if uncertainty remained after the clinical examination.Maternal blood and amniotic fluid samples were obtained at the time of admission before the administration of corticosteroids, antibiotics, or tocolytics. The performance of transabdominal amniocentesis to assess the intra-amniotic environment has been a part of the department's standard clinical management of women with PPROM. Women with PPROM were further managed based on amniotic fluid test results. Those with confirmed intra-amniotic inflammation received intravenous clarithromycin for seven days unless delivery occurred. Women without intra-amniotic inflammation were treated with intravenous benzylpenicillin  for seven days unless delivery occurred. Once the final results regarding microbial invasion of the amniotic cavity from cultivation and/or PCR were known, the attending clinician decided to modify the antibiotic therapy. Corticosteroids (betamethasone) were administered to those at gestational ages between 24 + 0 and 34 + 6 weeks to accelerate lung maturation. Tocolysis (atosiban) was used in those who developed regular uterine activity during the course of corticosteroid therapy or within 24 h after their administration.Women with proven intra-amniotic infection beyond the 28th gestational week were managed actively . The remaining women with PPROM were managed expectantly.After delivery, the placenta, fetal membranes, and umbilical cord were fixed in 10% neutral buffered formalin and sent for histopathological evaluation.This study was approved by the approved by the Institutional Review Board of the University Hospital Hradec Kralove . All experiments were performed in accordance with relevant guidelines and regulations. All participants in the study were Caucasian. Informed consent was obtained from all participants.34, cervical fluid samples35, and clinical and demographical data37 from some women from this cohort were used in our previous studies. Therefore, microbial results from amniotic fluid were shown, in part, in these publications37.Amniotic fluid samplesUreaplasma spp., Mycoplasma hominis, and Chlamydia trachomatis; 16S rRNA gene; and aerobic/anaerobic cultivation. The remaining amniotic fluid was further processed, and aliquots and pellets were frozen and used for research purposes. Details about amniotic fluid sampling were described in our previous publication20.During free-hand ultrasonography-guided amniocentesis, performed at the time of admission before administration of corticosteroids, antibiotics, or tocolytics, approximately 3 mL of amniotic fluid was aspirated and used for the assessment of IL-6 concentration; PCR for 38. The measurable range was 1.5–5000 pg/mL, which could be extended to 50,000 pg/mL with a tenfold dilution of the sample. The coefficients of variation for the inter-assay and intra-assay precisions were both < 10%.The concentration of IL-6 in the amniotic fluid was assessed using the automated electrochemiluminescence immunoassay method with the immuno-analyzer Cobas e602 C. trachomatis/Ureaplasma/M. hominis-FRT kit  was used to detect the DNA of Ureaplasma spp., M. hominis, and Ch. trachomatis using a single PCR tube for each fluid. The details have been described previously40. The level of Ureaplasma spp. (copies/mL) was determined using an absolute quantification technique that uses an external calibration curve. Plasmid DNA  was used to prepare the calibration curve.A commercial AmpliSens® 41. The details have been described previously40. The bacteria were then typed using the sequences obtained from BLAST® and SepsiTestTM BLAST20.Bacterial DNA was identified by PCR targeting the 16S rRNA using the following primers: 5-CCAGACTCCTACGGGAGGCAG-3 (V3 region) and 5-ACATTTCACAACACGAGC-GACGA-3 (V6 region)Gardnerella vaginalis selective medium, MacConkey agar, Neisseria-selective medium (modified Thayer–Martin medium), Sabouraud agar, and Schaedler anaerobe agar. The details have been described previously39.The amniotic fluid samples were cultured on Columbia agar with sheep's blood, 43.A maternal blood sample was obtained by venipuncture of the cubital vein and sent to the laboratory immediately following sampling to assess the CRP and WBC count. Details about maternal blood CRP and WBC count are described in our previous publications44. Histopathological examinations were performed by a single pathologist (HH) who was blinded to the clinical status of the women.Tissue samples were obtained from the placenta (at least two samples), fetal membranes , and umbilical cord , which were routinely processed and embedded in paraffin. Sections of the tissue blocks were stained with hematoxylin and eosin. The degree of neutrophil infiltration was evaluated separately in the free membranes (amnion and chorion-decidua), chorionic plate, and umbilical cord according to the criteria provided by Salafia et al.45.Maternal and perinatal medical records were reviewed by five investigators . Data regarding short-term neonatal morbidity were reviewed for all the newborns. Definitions of selected aspects of short-term neonatal morbidity have been described in our previous publicationsUreaplasma spp., M. hominis, C. trachomatis, or their combination; positivity for the 16S rRNA gene; positivity for aerobic/anaerobic cultivation of the amniotic fluid; or a combination of these parameters. Intra-amniotic inflammation was defined as an amniotic fluid with an IL-6 concentration of greater than or equal to 3000 pg/mL46. Colonization was defined as the presence microbial invasion of the amniotic cavity without intra-amniotic inflammation. Intra-amniotic infection was defined as the concurrent presence of microbial invasion of the amniotic cavity and intra-amniotic inflammation. Sterile intra-amniotic inflammation was defined as the presence of intra-amniotic inflammation without microbial invasion of the amniotic cavity. Negative amniotic fluid was defined as amniotic fluid without intra-amniotic inflammation and microbial invasion of the amniotic cavity. The intra-amniotic inflammatory response was characterized by the concentration of IL-6 in the amniotic fluid. Maternal inflammatory response was determined by the concentrations of CRP concentrations and WBC counts in maternal blood. HCA was diagnosed based on the histological grades 3–4 for the chorion-decidua, and/or grades 3–4 for the chorionic plate, and/or grades 1–4 for the umbilical cord, and/or grades 1–4 for the amnion44. Funisitis was diagnosed based on histological grades 1–4 for the umbilical cord44. Inflammation of the amnion was diagnosed based on histological grades 1–4 for the amnion44. Compound neonatal morbidity was defined as the need for intubation, and/or respiratory distress syndrome, and/or transient tachypnea of newborns, and/or bronchopulmonary dysplasia, and/or retinopathy of prematurity, and/or intraventricular hemorrhage, and/or necrotizing enterocolitis, and/or intestinal perforation, and/or early-onset sepsis, and/or late-onset sepsis, and/or neonatal death before hospital discharge20.Microbial invasion of the amniotic cavity was determined based on positive PCR analysis for U tests for continuous variables, as appropriate, and Fisher's exact or chi-square tests for categorical variables, as appropriate, and results were presented as median (interquartile range [IQR]) and number (%), respectively. The normality of the data was tested using the Anderson–Darling test. The loads of Ureaplasma spp. DNA in the amniotic fluid were not normally distributed; therefore, the non-parametric Mann–Whitney U test was used for the analyses. Kaplan–Meier survival curves were constructed, and a log-rank (Mantel-Cox) test was used to compare survival distributions among women with colonization, sterile intra-amniotic inflammation, and negative amniotic fluid. Spearman's partial correlation was used to adjust the results for gestational age at delivery. Differences were considered significant at P < 0.05. All p-values were determined using two-tailed tests, and all statistical analyses were performed using GraphPad Prism v8 for Mac OS X  and the Statistical Package for the Social Sciences (SPSS), version 28.0.0.0, for Windows .The demographic and clinical characteristics of the patients were compared using the non-parametric Kruskal–Wallis or Mann–Whitney Supplementary Information.


However, whether the combination of melphalan with busulfan (BUMEL) conditioning outperforms HDMEL remains controversy. Accordingly, a systematic review and meta-analysis was carried out to compare the outcomes of HDMEL and BUMEL-based conditioning regimens in newly diagnosed MM patients having undergone auto-HSCT.High-dose melphalan , overall survival (OS), optimal treatment response after auto-HSCT, duration of stem cell engraftment and incidence of toxic events between patients undergoing BUMEL-based and HDMEL conditioning regimens. Hazard ratio (HR), mean difference (MD) or odds ratio (OR) corresponding to 95% confidence interval (CI) were determined to estimate outcomes applying RevMan 5.4 software. Publication biases were assessed by performing Egger’s test and Begg’s test by Stata 15 software.Ten studies with a total of 2855 MM patients were covered in the current meta-analysis. The results of this study demonstrated that patients having received BUMEL-based regimen was correlated with longer PFS  but similar OS  compared with those having received HDMEL. The differences of best treatment response after auto-HSCT and duration of neutrophil or platelet engraftment did not have statistical significance between the two groups of patients. With respect to adverse effects, the patients in BUMEL-based group were less frequently subject to gastrointestinal toxicity while the patients in HDMEL group less often experienced mucositis and infection. No significant difference was observed in hepatic toxicity between the two groups of patients.In the present study, BUMEL-based conditioning was identified as a favorable regimen for a better PFS and equivalent OS as compared with HDMEL, which should be balanced against higher incidences of mucositis and infection. BUMEL-based conditioning is likely to act as an alternative strategy to more effectively improve auto-HSCT outcomes in MM.The online version contains supplementary material available at 10.1186/s12935-021-02313-z. Multiple myeloma (MM) refers to a hematological malignancy, as manifested by excessively proliferated monoclonal plasma cells in the bone marrow, thereby causing renal dysfunction, bone marrow failure and bone destruction. Over the past two decades, the incidence of MM has increased globally by 126% whereas the age-standardized mortality rate has been steadily falling . MoreoveEven so, the disease remains incurable, and considerable patients are eventually subject to disease relapse. Several approaches concentrating on different stages of MM treatment have been developed to down-regulate the incidence of disease progression, including improving the induction of chemotherapy prior to auto-HSCT by combining novel agents –4, and a2) has been considered as the universal standard conditioning regimen for auto-HSCT in MM , duration of stem cell engraftment and incidence of toxic effects between the two groups of patients.Two reviewers (F Gao and M Zhang) independently extracted the data and assessed the quality of included studies. The following information from the respective study was summarized: (1) first author, (2) year of publication, (3) country, (4) induction treatment regimens, (5) conditioning regimens, (6) number of patients in each arm of study, (7) follow-up time, (8) number of patients having received maintenance treatment, and (9) primary and secondary outcomes of current study.The quality of retrospective studies was assessed by adopting the Methodological Index for Non-Randomized Studies (MINORS) scale . Given tHazard ratio (HR) corresponding to 95% confidence interval (CI) was adopted to assess OS and PFS. If the results of univariate and multivariate analyses were reported in the included studies, the latter will be applied to the meta-analysis. If HRs and 95% CIs were not available from the original study, Kaplan–Meier curves of the included studies were read and re-analyzed by software Engauge digitizer. HRs and 95% CIs were indirectly calculated from Kaplan–Meier curve using Tierney’s methods . Mean di2. Patients in the BUMEL-based group received conditioning regimen of busulfan plus melphalan in nine studies while received busulfan, melphalan plus bortezomib in one study. All the included studies had reliable quality as indicated by final scores of MINORS scale ranged from 16 to 19 for nine retrospective studies [Figure  studies , 25–31 . Meta-analysis revealed that MM patients having received BUMEL-based conditioning regimen had significantly favorable PFS when compared with those having received HDMEL alone .A total of 9 studies were eligible for the assessment of OS. No significant difference was identified in OS between the two groups of patients . The result of meta-analysis indicated that no difference of the best response after auto-HSCT was found between patients in the BUMEL-based group and the HDMEL group  were also comprehensively compared between patients in the BUMEL-based group and the HDMEL group. The results indicated that gastrointestinal toxicity was significantly less frequently observed in the BUMEL-based group than that in the HDMEL group  in MM patients significantly increased toxicity , without clear survival advantage over melphalan at 200 mg/m2 despite the improvement of treatment response [2. In the current study, the most noticeable finding was the longer PFS found in MM patients having received conditioning of BUMEL-based as compared with those having received HDMEL 200 mg/m2, which might be explained by the synergistic effect of the combination of busulfan and melphalan [As indicated from an existing study, improving pre-transplantation conditioning chemotherapy by applying higher doses of melphalan  [2 group, which might have a critical impact on OS. Additionally, the different percentage of patients having received maintenance treatment and different maintenance treatment regimens between the two groups of patients might also have significant influence on OS.The favorable PFS identified in the BUMEL-based group did not translate into the advantage of OS. A possible interpretation referred to the subsequent lines of rescue treatment after relapse. Lahuerta JJ, et al. reported that the use of novel agents as salvage therapy showed an independent positive prognostic effect on OS regarding the use of conventional chemotherapy at relapse/progression . Notablyse (VOD) . This diIn terms of toxicity, we observed that the incidence of gastrointestinal events was significantly lower in the BUMEL-based group as compared with that in the HDMEL group, which might be explained by the reduced dose of melphalan in the BUMEL-based group as gastrointestinal toxicity, such as diarrhea, nausea, and vomiting, was one of the most common adverse effects exerted by melphalan. However, infection and mucositis were more frequently identified among BUMEL-based recipients for higher intensity of the combined alkylating agent conditioning regimen of busulfan and melphalan. Nevertheless, the mentioned adverse events were manageable and did not extend to transplant-related mortality. Besides, the application of intravenous busulfan with pharmacokinetic dose adjustment could be conducive to reducing the incidence of VOD and other hepatic toxicity. Thus, the frequency of hepatic event was similar between the two conditioning groups in the existing study. Given the toxicity of melphalan and busulfan, delivery melphalan and busulfan simultaneously or separately by polymer nanoparticles may be a promising pharmaceutical design in clinic to reduce the adverse effect and further improve the efficacy of BUMEL conditioning regimen in the future .To the best of the authors’ knowledge, this study is the first meta-analysis systematically assessing clinical outcomes between BUMEL-based and HDMEL as the conditioning regimen before auto-HCT for MM. We are confident that the results of this study were reliable, which was supported by large sample size, moderate to high methodological quality of included studies, low heterogeneity, and no publication bias. However, there were several limitations in this study. First, when evaluating OS or PFS, HR and 95% CI in some of individual studies were not available from the original studies, hence they were indirectly calculated from Kaplan-Meier curve. Second, minimal residual disease (MRD) testing has been widely used as a robust method for assessment of clinical efficacy of certain treatment regimen in MM , 38. HowThe present meta-analysis revealed the high anti-myeloma activity of BUMEL-based conditioning prior to auto-HSCT in MM, which could be correlated with superior PFS and comparable OS compared with HDMEL conditioning though a higher incidence of manageable adverse events was found. BUMEL-based conditioning regimen is likely to act as an appropriate strategy to further improve long-tern outcomes in MM patients. Meanwhile, we should also pay attention to toxicity of BUMEL-based conditioning regimen and appropriate management is required. In view of the existing limitations of the current study, a well-designed prospective study with large cohorts should be conducted to clarify the mentioned issues in the future.Additional file 1: Table S1. PRISMA 2020 Checklist. Table S2. The details of the search strategy. Additional file 2: Figure S1. MINORS Scale to assess the study quality.


Breast reconstruction is most commonly performed using implant-based reconstruction. Patients with subpectoral implant placement with or without latissimus dorsi (LD) muscle coverage can experience muscle pain and animation deformity. Due to minimal literature describing the use of botulinum toxin (BTX-A) treatment for these side effects from implant-based reconstruction, we report our outcomes.A retrospective chart review of breast reconstructive patients for a single surgeon was performed. Patients who underwent BTX-A injection for muscular pain, spasm, or animation deformity were identified and outcomes reviewed. They were also stratified based on radiation treatment and type of muscle flap used.Eleven patients were identified who had a submuscular pectoralis pocket and/or a pedicled latissimus dorsi flap. Nineteen breasts were treated. The average amount of time from the patient’s last surgery to BTX-A injection was 11.2 months. 25–100 units were used per injection with an average of 60 units. Non-irradiated patients had signifycantly lower post-injection capsular contracture Baker grades and significantly lower amounts of BTX-A were injected. Patients who had both pectoralis major muscle and LD implant-reconstruction were significantly less likely to have improvement in pain/tightness. Most patients reported improvement or resolution of their pain and/or animation deformities.Implant-based reconstruction using the pectoralis major and/or LD muscles can be plagued with muscular pain, spasm, and animation deformities. The use of BTX-A is a diagnostic and therapeutic modality for these post-breast reconstruction patients with most patients having resolution of symptoms without the need for additional surgery. It inhibits acetylcholine and substance P release causing muscle paralysis with a less clear mechanism of action causing decreased pain , 2. One Animation deformity can range in severity, from slight discomfort and embarrassment to profound pain and restriction of physical function. It is estimated that at least 75% of subpectoral patients experience some degree of AD, and it is a common cause for revision surgery, with approximately 28% of patients pursuing reoperation . Pectora2.A retrospective chart review of patients undergoing breast reconstruction with a single surgeon was performed. Patients who underwent implant-based reconstruction with supplemental use of the pectoralis major muscle and latissimus dorsi muscle were identified. Those who had persistent post-operative muscular pain, contraction, or animation deformity and underwent BTX-A injection for these symptoms and signs were identified. Their demographic information was reviewed along with the details surrounding the BTX-A injection , as well as the patients’ outcomes. Improvement of muscular pain and animation deformity were the primary endpoints used to determine the need for repeat injection or surgery. This was determined based on the patient’s report, as well as on physical exam. The study was approved by the institutional review board.2.1Botulinum toxin type-A injection was performed in an outpatient clinic without any sedation or local anesthetic. The freeze-dried 100 unit BTX-A vial was reconstituted with 5 mL of sterile sodium chloride (0.9%). The location of the patient’s pain and/or muscle tightness was identified at rest and with activation by having the patient place their fists on their hips while adducting and internally rotating their arms. Occasionally focal areas of muscle spasms can be seen visually, as well as by palpation. In addition, the patient’s reported sites of pain and/or tightness were marked to provide targeted injection including the pectoralis major muscle’s origin . The amoThe areas previously marked during the exam identifies the areas of injection, which are prepped with alcohol. The pectoralis major muscle is palpated under the skin and confirmed with muscle activation. The skin and muscle at the site of the injection is pinched between fingers and the implant is displaced away from the injection site. The injection is performed using the entire length of the 30G ¼ inch needle into the tissue isolated between the fingers. Awareness of the injection location and length of the injecting needle, as well as displacement of the implant was performed to avoid injury to the underlying implant. Of note, the use of BTX-A for these purposes is off-label and not yet approved by the FDA.2.2All statistical analyses were completed using Stata v.15 . The Shapiro-Wilk test was used to determine whether continuous variables were normally distributed. Clinical data were compared between cohorts using Kruskal-Wallis and Fisher’s Exact analyses. The two-tailed threshold for statistical significance was set at an alpha value of 0.05.3.From 2012 to 2019, eleven women meeting inclusion criteria were identified. The average age was 51.5 years (range 34–73 years old) with the majority of patients  having a diagnosis of breast cancer . All patPatient response to the BTX-A injections were assessed during their post-operative visits. The average length of follow-up was 8.5 months (range 8 days to 24.5 months) from the time of the patient’s last BTX-A injection. Two patients were lost to follow up. The majority of remaining patients  reported improvement or resolution of their pain and/or animation deformities, as well as having softening of their capsule contracture to grade 1–2 on exam. Three patients required 2 or more BTX-A injections and 1 patient had denervation of bilateral pectoral nerves due to the severity of the animation deformity that had improved, but recurred, with BTX-A. No adverse complications were associated with BTX-A use including injection site infection, generalized muscle weakness, breathing difficulties or hypersensitivity reactions. When stratifying patients by their history of radiation therapy, 4 patients were identified as having undergone radiation . All of In addition, when analyzing the effects by breast, non-irradiated breasts received significantly lower amounts of BTX-A than irradiated breasts . Patient4.Breast reconstruction is becoming increasingly widespread after mastectomy. The most common approach is implant-based reconstruction with the majority of patients undergoing subpectoral placement versus prepectoral placement , 21, 22.In general, the literature on the use of BTX-A in breast reconstruction is conflicting. A nonrandomized prospective study of 48 patients (22 patients receiving and 26 patients not receiving BTX-A infiltration at the time of mastectomy) reported a significant decrease in post-operative pain and decreased narcotic requirement in the study group . A randoOur results showed that non-irradiated patients had significantly lower Baker grades of capsular contracture post-injection compared to irradiated patients. The non-irradiated breasts also were significantly more likely to demonstrate improvement in symptoms of pain/tightness. This suggests that BTX-A is more efficacious in treating capsular contracture among non-irradiated patients. In addition, non-irradiated patients received a significantly lower amount of BTX-A. These results indicate that the skin and soft tissue changes after radiation may have a negative impact on the effectiveness of BTX-A. When comparing the type of muscle coverage using pectoralis major muscle and/or latissimus dorsi muscle, the combined use of these muscles were significantly less likely to have an improvement in pain/tightness symptoms after injection and a higher post-injection pain score. With muscle being used from two sites, it is not surprising that patients would be more symptomatic as it is likely there is an additive effect when combining both muscles. The limitations associated with this study are its small sample size, retrospective nature, and variable length of follow-up. In addition, while the use of BTX-A has been reported in implant-based reconstruction, a specific methodology still needs to be established. Approaches have ranged from the number of units used to the number and specific locations of the injections, and the optimal dose of BTX-A has yet to be determined.5.The use of BTX-A can be a diagnostic and therapeutic modality for implant-based breast reconstruction patients who experience muscular pain, spasm, and animation deformities due to the manipulation of their pectoralis major and/or latissimus dorsi muscles. This study supplements the available literature that generally supports the use of BTX-A for these symptoms and may negate the need for additional surgery in these patients. Therefore, botulinum toxin A is a promising first-line therapy for post-implant breast reconstruction patients experiencing muscular pain and dysfunction.