Severe upper limb paresis can represent an immense burden for stroke survivors. Given the rising prevalence of stroke, restoration of severe upper limb motor impairment remains a major challenge for rehabilitation medicine because effective treatment strategies are lacking. Commonly applied interventions in Germany, such as mirror therapy and impairment-oriented training, are limited in efficacy, demanding for new strategies to be found. By translating brain signals into control commands of external devices, brain-computer interfaces (BCIs) and brain-machine interfaces (BMIs) represent promising, neurotechnology-based alternatives for stroke patients with highly restricted arm and hand function. In this mini-review, we outline perspectives on how BCI-based therapy can be integrated into the different stages of neurorehabilitation in Germany to meet a long-term treatment approach: We found that it is most appropriate to start therapy with BCI-based neurofeedback immediately after early rehabilitation. BCI-driven functional electrical stimulation (FES) and BMI robotic therapy are well suited for subsequent post hospital curative treatment in the subacute stage. BCI-based hand exoskeleton training can be continued within outpatient occupational therapy to further improve hand function and address motivational issues in chronic stroke patients. Once the rehabilitation potential is exhausted, BCI technology can be used to drive assistive devices to compensate for impaired function. However, there are several challenges yet to overcome before such long-term treatment strategies can be implemented within broad clinical application: 1. developing reliable BCI systems with better usability; 2. conducting more research to improve BCI training paradigms and 3. establishing reliable methods to identify suitable patients. Stroke is a leading cause for long-term disability and often results in poor quality of life . In 2017Upper limb impairment occurs in approximately 80% of stroke survivors . Here, tConstraint-induced movement training (CIMT) represents an effective motor intervention in upper limb rehabilitation –13. HoweWhile there are well-evidenced motor interventions for mild paralysis, successful rehabilitation of severe upper limb paresis is still an unsolved problem: For classical physiotherapy, evidence of efficacy is not convincing , 22, 23.Brain computer interfaces (BCIs) use the modulation of neurometabolic or neuroelectric signals to control external devices . By analBCI technology addresses important physiological fundamentals of neurorehabilitation. Based on Hebbian learning principles, such systems can induce neuroplasticity by effectively coupling efferent brains signals to afferent input  . This waAlthough BCI-based approaches appear to be promising for upper limb rehabilitation, sufficient evidence for broad clinical application is still lacking and more research is needed. However, conducting studies according to principles of evidence-based medicine (EBM) is often difficult due to the heterogeneity of stroke patients. Further, BCI-based motor therapy involves far more variables than just dose and timing, and double blinding is difficult to implement. Therefore, Coscia et al.  suggest Thanks to its various applications, BCI-based therapy can be embedded in many stages of stroke rehabilitation. Here, based on a literature review, we derived the best strategies to facilitate and accelerate the integration of BCI-based therapy into the German process of neurorehabilitation. We include studies that assess the impact of BCI-based interventions on motor recovery of stroke patients with severe upper limb paresis, either in the subacute (<6 months) or chronic stage (>6 months). Study outcomes were either functional scores and/or neurophysiological parameters. Case studies and studies with small sample size (<10 participants) were not included. In Germany, the process of neurological rehabilitation is divided into 6 phases (A to F): Phase A represents the emergency care on a stroke unit, followed by phase B which is equivalent to early rehabilitation. In Phase C, patients can actively participate in rehabilitation interventions, however, there is still high demand for medical treatment. In Phase D, patients receive medical rehabilitation in specialized rehabilitation centers (Phase D), which corresponds to the concept of post hospital curative treatment. In Phase E, outpatient occupational reintegration is pursued and, in case long-time care is necessary, Phase F is initiated to support and maintain function .In acute care and early rehabilitation (Phase A and B), BCI-based applications often cannot be applied due to patient's reduced vigilance and impaired cooperativity . HoweverAlthough there are no RCTs comparing early vs. late onset, a general recommendation is that the earlier motor interventions start, the better . Thus, BThere are good reasons to continue BCI therapy in chronic stroke patients that already showed some degree of motor recovery. In patients with initial hemiparesis but returning arm control, EEG-based hand exoskeletons can be used to restore hand function. It was shown that the application of a BCI-driven hand exoskeleton in chronic stroke patients reduces complete finger paralysis , improveIn case no sufficient improvement can be achieved, nursing or family care (Phase F) is often unavoidable for patients with remaining severe upper limb impairment. Although the rehabilitative potential might be exhausted in these cases, the BCI approach can be used to compensate for lost function by controlling assistive devices, e.g., exoskeletons or robotic prostheses (assistive BCIs). Bundy et al.  have demAttaching and calibrating BCI systems currently requires the support of specially trained personnel and takes considerable amount of time . Since tBCIs can fail to detect modulation of brain activity in up to 20% of individuals . While sFor motor interventions to be most effective, it has been suggested to move from a “one-fits-all” approach toward customized rehabilitative interventions . In thisWith its capability to advance motor recovery, BCI-based motor interventions provide a good argument to be implemented in stroke rehabilitation in Germany. BCI-based neurofeedback training is well suited to promote neural and functional recovery in early rehabilitation. BCI-based motor interventions showed efficiency in both subacute and chronic stroke patients, making them suitable for post hospital curative treatment and outpatient occupational therapy. Once a plateau in motor recovery is reached, assistive BCI-based devices can be applied in a home setting to improve quality of life. Put together, BCIs represent a promising approach for long-term treatment of severe upper limb paresis in stroke patients. Nevertheless, there are still some obstacles to overcome before BCI-based therapies can be applied in routine clinical practice.CA and SRS conceived the topic. CA conceptualized the manuscript. CA took the lead in writing the manuscript and was supported by AC, MV, VH, and SRS. All authors provided critical feedback and helped shape the research idea and manuscript, reviewed the results, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Various living organisms have proven to influence human health significantly, either in a commensal or pathogenic manner. Harnessing the creatures may remarkably improve human healthcare and cure the intractable illness that is challenged using traditional drugs or surgical approaches. However, issues including limited biocompatibility, poor biosafety, inconvenience for personal handling, and low patient compliance greatly hinder the biomedical and clinical applications of living organisms when adopting them for disease treatment. Microneedle arrays (MNAs), emerging as a promising candidate of biomedical devices with the functional diversity and minimal invasion, have exhibited great potential in the treatment of a broad spectrum of diseases, which is expected to improve organism-based therapies. In this review, we systemically summarize the technologies employed for the integration of MNAs with specific living organisms including diverse viruses, bacteria, mammal cells and so on. Moreover, their applications such as vaccination, anti-infection, tumor therapy and tissue repairing are well illustrated. Challenges faced by current strategies, and the perspectives of integrating more living organisms, adopting smarter materials, and developing more advanced technologies in MNAs for future personalized and point-of-care medicine, are also discussed. It is believed that the combination of living organisms with functional MNAs would hold great promise in the near future due to the advantages of both biological and artificial species. The individual needle on MNAs is well-shaped  with the length ranging from 200 to 1500 μm, and the diameter is around 250 to 600 μm e.g., interstitial fluid or blood detection based on chemical, electrochemical or immune strategies) and specific drug delivery  With the development of novel materials and micromachining techniques, the emerging microneedle technology has become a promising candidate to treat various diseases in a minimally invasive manner without sophisticated equipment or specific professionals Figure 1). Initially, we briefly introduce the specific classification and materials of MNAs used to integrate organisms. Subsequently, detailed integration strategies using the living organism and MNAs, and their corresponding applications for drug delivery and disease treatment are categorized according to the species of the organisms . Finally, we highlight the challenges faced by current strategies, and the perspectives including more living organisms, smarter materials and advanced novel technologies are discussed in-depth.In this review, we systemically summarize the combinative systems of MNAs and living organisms, with specific biological species including diverse viruses, bacteria, mammal cells, and so on. Moreover, their applications such as vaccination, anti-infection, tumor therapy, and tissue repairing are well illustrated (Strategies based on organism-MNA combination are mainly divided into two categories: (1) Directly applying living organisms into tissues/organs to induce repairing effects or the immune response. (2) Utilizing the secretion of living organisms to mediate metabolism or eradicate pathological cells/bacteria. Given the different cargoes (living species or their secretion) that needed to be delivered, MNA forms with distinct constitutions and working mechanisms are optimized. Moreover, the material selection for organism-integrated MNA fabrication should include the following features: (1) Suitable stiffness to ensure tissue penetration. (2) Excellent biocompatibility with minimal damage to the hosts and the integrated organisms. (3) Sufficient loading capacity of therapeutic agents for disease treatment.Currently, there are mainly four types of MNAs, including bulk, coated, dissolving and insoluble MNAs. Each type of these MNAs adopts specific materials for fabrication and has its own unique delivery profile e.g., stainless steel), silicon and stiff polymers ) with or without channels inside the MNAs. They are able to form micro-holes on the surface of target tissues to facilitate the therapeutic agents' transport. Among all types of MNAs, bulk MNAs display the highest mechanical strength, ensuring successful insertion and thus guaranteeing the delivery efficiency. Therefore they are widely employed for virus/bacteria-based vaccination.Bulk MNAs are mainly made of stiff materials such as metal  that encapsulate therapeutic agents inside. After inserting into tissues, the whole microneedles are dissolved in ISF and release the payloads. In order to trigger ignorable immunogenicity or toxicity when degraded, and to effectively maintain the viability of organisms in MNAs, natural extracts like carboxymethyl cellulose (CMC), hyaluronic acid (HA) and saccharides , or synthesized polymers such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) and poly(ethylene glycol) (PEG) with benign biocompatibility are commonly employed e.g., spontaneously responding to blood glucose variation) Differently, coated and dissolving MNAs would dissolve into target tissues to release the encapsulated organisms after penetration. For coated MNAs, agents such as drugs or regulatory factors are directly deposited on the surface of MNA tips, which enables well mixture of the payload with interstitial fluid (ISF) to improve their diffusion into target locations. Meanwhile, dissolving MNAs are made of dissolvable materials  and simple structures (only including nucleic acids and proteins), the viruses have been recognized as highly dangerous organisms given their high pathogenicity and infectivity e.g., human adenovirus serotype 5 (AdHu5) and chimpanzee serotype 63 (ChAd63)) have been confirmed to be safe and immunogenic in human clinical trials Recombinant viruses, which are alive but non-replicating, can be gene-edited by inserting exogenous genes that encode antigens of malaria, influenza, tuberculosis, HIV, and so on, serving as effective vaccines et al. developed silicon MNAs with different dimension-related parameters, and compared their influence on the magnitude and memory of vaccine-induced CD8+ T cells et al. from the same research group also delivered an adenovirus-vectored malaria vaccine using the identical bulk MNAs, achieving corresponding results et al. investigated a spray-coating technique to load vaccines onto MNAs et al. adopted sodium carboxymethyl cellulose (Na-CMC) to fabricate a dissolving MNA containing AdHu5-vectored vaccine, which effectively exerted functions for at least one month. Their work also revealed the critical roles of different immune cells performed during the MNA vaccination  MNA technology has been widely applied for recombinant virus delivery to induce an intense immune response, and bulk MNAs are usually utilized given their excellent delivery efficiency. Moore et al. engineered phase-transition MNAs loaded with adeno-associated virus (AAV) to deliver genes of vascular endothelial growth factor (VEGF) for cardiovascular disorder treatment  MNAs are also utilized to deliver recombinant viruses as gene carriers toward treating diseases based on gene therapy. Ye et al. engineered MNAs to administer measles and rubella (MR) vaccine in rhesus macaques models  Besides recombinant viruses, natural viruses themselves have been broadly employed for infectious disease prevention and tumor treatment. Because replication competence and viral virulence do not frequently correlate with immunological robustness, live-attenuated viruses could also serve as potential vaccines. After attenuation, virus toxicity would decrease but their immunogenicity is reserved so that they can still trigger the immune response effectively e.g., oncolytic viruses) et al.  In addition to vaccines, viruses could also be applied to treat tumors (Table 2).It is well known that bacteria have shown a complicated relationship with human health for years et al. employed a silicon MNA on an in vitro model (excised porcine skin) to assess the infection potential of lesions compared with hypodermic needles i.e., C. albicans, P. aeruginosa and S. epidermidis) on the surface of skin models and punctured the skin with hypodermic needles or MNAs. Subsequently, the number of bacteria in the skin was calculated to evaluate the possibility that organisms traversed the MNA-induced channels. Results obviously demonstrated that MNAs would not result in severe local or systemic infections, and thus validating the minimal invasion and safety of MNAs. Cui et al. obtained similar conclusions using a commercial MNA roller (Dermaroller®) made of metal  et al. tested them in mice models  F. novicida, a kind of bacteria that caused tularemia, was adhered onto a bulk MNA made of poly(L-lactide acid) (PLLA), and applied onto C57BL/6 mice. Successful bacteria deposition was observed, and survival rate and tissue response  such as T-cell depletion and inflammatory cytokine levels were monitored. Their results evidently showed that MNAs could effectively deliver bacteria into the skin to regulate immune responses in vivo.Living bacteria can be applied to evaluate the damage of the skin triggered by bulk MNAs. Donnelly et al. invented a dissolving MNA that contained caves near the bases of needles to store live-attenuated Bacille Calmette-Guerin (BCG) bacillus as a vaccine bacillus could be stored for at least 90 days at room temperature, and was able to induce desirable immune protection, validated by intracellular cytokine productions and humoral responses in mice models. This MNA-based strategy exhibited similar inoculation efficiency compared to traditional intramuscular injection, while it did not require any medical professionals/equipment and cold-chain transportation, therefore facilitating popularization.With advances of biomaterials that could endow MNAs with smart functions like stimuli-responsiveness and sustained release, MNAs could be applied to significantly enhance bacteria-based therapies e.g., fungal infection). Xie et al. encapsulated lactobacillus in the dissolvable sodium carboxymethyl cellulose and subsequently micromolded MNAs, followed by local delivery into the skin . Results clearly showed that the probiotics inside the MNAs were kept alive at 4 ℃ for at least 21 days, and they could continuously produce lactic acid for 2 hours, and would not cause subcutaneous inflammation.Other than being used as vaccines, bacteria, especially those probiotics, are also integrated into MNAs to treat skin diseases  Given that some bacterial secretion may act as new agents to treat diseases Recently, cell-based therapies emerge as a fast-growing field, and present great potential in tissue/organ repairing/regeneration and cancer treatment Table 3. Assisted by abundant functional materials, MNAs are able to encapsulate versatile cells and deliver them to various tissues, and therefore benefit the advance of point-of-care cell therapies. Based on the locations of the cells in the MNAs, these systems can be divided into cell-delivery MNAs and secretion-delivery MNAs.Typical MNAs that could be employed for cell delivery are summarized in et al. tried to deliver skin cells  using bulk polymethyl methacrylate (PMMA) MNAs, which were successfully applied onto in vitro model (collagen hydrogel) 2 or Si3N4Bulk MNAs have been attempted to deliver mammal cells by creating microchannels on the surfaces of target tissues. Huang et al. utilized dissolving MNAs made of hyaluronate (HA)/polyvinyl pyrrolidone (PVP)/maltose to deliver cancer cells into mice models for tumor grafting  et al. achieved localized MSC therapy using a similar cell-based dissolving MNA strategy  Compared to bulk MNAs, the utilization of biocompatible materials may provide high tunability to mediate cell behaviors. In these kinds of MNAs, cells were frequently encapsulated in needle tips and then were directly released in the tissues after penetration. For example, Wanichwecharungruang via MNAs was developed by Xu et al. using a cryogenic method In vivo models validated that cells delivered by those cryomicroneedles remarkably retained their viability (ranging from 21.4% to 53.1%) and proliferative capability. In mice models with subcutaneous melanoma tumors, the delivery of ovalbumin-pulsed dendritic cells via the cryomicroneedles elicited higher antigen-specific immune responses and led to slower tumor growth compared to intravenous and subcutaneous injections of the cells. These biocompatible cryomicroneedles exhibited great potential to facilitate minimally invasive cell delivery and improve patients' compliance for a range of cell therapies  into the pathological tissues to treat corresponding diseases. In those MNAs, functional cells are frequently encapsulated inside the base parts or on their surfaces, and thus are protected by cross-linked insoluble hydrogels against the host immune system. The needle tips of the MNAs create “microchannels” on the surfaces of target tissues and then the secreted therapeutic agents diffused through the microchannels to the pathological locations. Due to the continuously viability maintenance of the encapsulated cells, the cellular secretion persistently treats the lesions for a long time.Other typical cell-based MNAs deliver not cells but their secretion  Considering the limitations of direct cell implantation for diabetes treatment such as host immunological rejection, Gu et al. developed polymeric MNAs integrated with cardiac stromal cells (CSCs) for therapeutic heart regeneration  et al. which consisted of three layers: a drug-loaded MNA (bottom layer), parallel-aligned carbon nanotube (CNT) conductive (middle layer), and a methacrylated gelatin (GelMA) hydrogel scaffold (upper layer) MNAs can provide ingenious strategies to improve cell-based therapies by preventing immune response and extending treatment periods, for example, in the field of curing heart diseases. Heart-derived cardiac stem/stromal cells can secrete regeneration factors to promote endogenous repair of the injured myocardium. However, the low cell viability by injection and the poor fusion with the host myocardium posed unique challenges for their applications. Cheng Given the advantages of minimal invasion and easy operation, MNA technology has shown great potential in the fields of point-of-care drug administration, and convenient personalized diagnosis and therapies. Besides sampling biomarkers and delivering therapeutic agents, integrating with living organisms endows MNAs with versatile abilities from vaccination to cell-based therapies. Viruses, bacteria and mammal cells, whose size finely matches with micro-scaled MNAs, are ideal organisms employed in MNAs, showing high promise for either organism or secretion delivery. Those MNAs have been applied in various fields including but not limited to triggering adaptive immunity, treating infections or tumors, and repairing tissues, which provides convenient alternatives without professionals/equipment compared to traditional therapies based on drugs or surgeries.Although it has exhibited great potential, the integration of living organisms in MNAs still faces several challenges that limit their development:e.g., saccharides) have been adopted for long-term storage of virus-integrated MNAs, how to maintain the viability of more complicated organisms such as bacteria or cells in MNAs remains challenging. Recently, specific hydrogels or cryogenic reagents are under investigation, but they frequently require specific storage conditions of liquid nitrogen or humid incubators, which conflicts with the purposes of using MNAs as ordinary and convenient therapeutic tools in daily life.(1) Maintaining bioactivity or viability of the integrated living organisms. Despite that various adjuvants , and the infection or tumorigenesis will happen if their proliferation is out of control, posing risks to the host's health. Possible mutations of those evolving organisms during their fast growth, under a pathological environment or caused by implanted materials, might influence the organism's behaviors such as proliferation, differentiation, and so on e.g., tumorigenesis due to gene mutation for cells). And the fast volume increase of these organisms as individual or group finally break the limitation of MNA structures, leading to their escape into the body and causing physical damages of the located tissues, excessive immune/inflammatory response or even death.(2) The biosafety of inducing living organisms into human beings. Since that MNAs either insert or directly implant into tissues, the contact between organisms and the human body lasts a period of time, either in a direct manner or separated by hydrogels. Therefore, the immunogenicity and the biosafety of those encapsulated organisms have to be considered. Certain organisms possess the ability of fast proliferation ((3) Limited species of organisms and few locations for MNA application. Currently, only limited viruses, bacteria and cells are integrated with MNAs due to a lack of effective strategies and materials to hold organisms or regulate their behaviors. Moreover, current applications of MNAs are mainly restricted to the skin and heart tissues. Lack of feasible and practicable application or adhesion manners is the main reason that hinders the development of MNAs on other tissues.In order to face the aforementioned challenges, attempts that devoted into following aspects are expected to further benefit human healthcare in a convenient, low-cost and point-of-care manner:Considering the versatile functions of extensive organisms and abundant biological tools developed to modify them, new species, besides the aforementioned viruses, bacteria and mammal cells, need to be tested in MNAs to seek novel strategies toward improving healthcare and treating diseases. For example, oxygen-producing algae can be utilized to remiss ischemic diseases that happened in the brain/heart Moreover, certain living creatures that possess immunogenicity could be applied to “train” the innate immune system to prevent various diseases. For instance, studies demonstrate that malaria infection can counteract tumor immunosuppressive microenvironment and inhibit the angiogenesis, growth and metastasis of tumors, which has been preliminarily validated by experiments on mice and human beings 2 or urea, and synthesize saccharides to supply nutrition to the target tissue. Furthermore, with the development of tissue engineering, many other human cells  are anticipated to maintain viability inside MNAs, and then implanted to target tissues for metabolic waste clearance in a well regulatory profile.In addition to the living species mentioned above, more organisms related to nutrition delivery and waste clearance could act as new candidates in MNA-biosystem combinations. For example, engineered bacteria/microalgae are expected to intake wastes such as COe.g., metal and silicon), abundant functional biomaterials, especially various natural or artificial polymers, have proven to significantly improve the performance of MNAs. Biocompatible materials have demonstrated the success of integrating living organisms with satisfactory viability inside MNAs by providing suitable microenvironments, for example, the adjuvant of saccharides is usually adopted to keep the virus stability when loading them into MNAs for vaccine inoculation. Biocompatible hydrogel  is mainly employed to encapsulate bacteria. During mammal cell encapsulation, the largest challenge is to maintain their viability in MNAs given the vulnerability of cells in vitro. One potential solution is to adopt special materials that are similar to the extracellular matrix for MNA fabrication, such as gelatin-methacryloyl (GelMA). Other methods focus on modifying reagents that are prepared for live cell storage at the low temperature, for example, the commonly-used cryogenic medium of dimethyl sulfoxide (DMSO) is incorporated with sucrose to balance between maintaining cell viability and realizing feasible MNA fabrication. What is more, new functionalized materials have drawn increasing attention in this field: (1) Besides biocompatibility, the newly-developed materials are expected to offer continuous nutrition supply, facilitating long-term creatures storage under room temperature or in a refrigerator. For example, extracts from silk (sericin and fibroin) are demonstrated to have a variety of biological activities for promoting cellular proliferation and inhibiting apoptosis, and thus benefit tissue regeneration e.g., gelatin, collagen, polyethylene glycol, poly(lactic acid) and so on) through covalent bonds and are able to direct cell behaviors Material science plays a significant role in MNA fabrication. Compared to traditional stiff materials with relatively sole functions . With the advances of interventional medicine techniques, specific catheters can go through natural lumens  and enable operations such as visualization, biopsy or minimally invasive surgery Since that MNA insertion requires extra pressure using applicators or thumb-pressing, current MNA-based systems are mainly limited to the skin and heart (during surgery). Therefore, developing new strategies based on MNA is urgently demanded. For instance, when being integrated with self-triggered spring e.g., quantum dots and indocyanine green) inside MNAs, and thus facilitate visualization of pathological lesions as well as accurate medical recordkeeping e.g., DNA and RNA) With the development of biomedical technologies, MNA functionalization would be significantly enhanced to extend their applications. For instance, molecular imaging can be integrated with MNAs by encapsulating proper imaging agents  to exert functions, the organism behaviors are expected to be finely tuned, indicating a highly regulatory fashion. When integrating with these techniques, the biggest advantage that MNAs can offer is the fast, convenient, and low-cost biomedical implementation without the assistance from professionals, which greatly benefits point-of-care diagnosis and therapies in a personal manner beyond hospitals.Recently, the technology of wearable electronics has grown pretty fast and shown promising potential in monitoring the physical/physiological status of the human body e.g., drugs or surgeries). With further development of biological, materials and engineering technologies, it is believed that the combination of living organisms with smart microneedle patches would hold great promise in the near future due to the advantages of both natural and artificial species.Overall, microneedle arrays integrated with living organisms have exhibited great potential in the fields of point-of-care drug administration and personalized therapies in specific diseases. Those MNAs have proven to play a significant role in vaccination and cell therapy, especially for diseases related to immune disorders, infections, tumors, tissue damage, and so on, which are difficult to treat by traditional therapies (


Stem cells possess regenerative powers and multidirectional differentiation potential and play an important role in disease treatment and basic medical research. Urine-derived stem cells (USCs) represent a newly discovered type of stem cell with biological characteristics similar to those of mesenchymal stromal cells (MSCs), including their doubling time and immunophenotype. USCs are noninvasive and can be readily obtained from voided urine and steadily cultured. Based on advances in this field, USCs and their secretions have increasingly emerged as ideal sources. USCs may play regulatory roles in the cellular immune system, oxidative stress, revascularization, apoptosis and autophagy. This review summarizes the applications of USCs in tissue regeneration and various disease treatments. Furthermore, by analysing their limitations, we anticipate the development of more feasible therapeutic strategies to promote USC-based individualized treatment. Stem cells, including embryonic stem cells (ESCs), adult stem cells (ASCs) and induced pluripotent stem cells (iPSCs), have increasingly emerged as research hot spots due to their roles in sustaining tissue homeostasis and regeneration and promoting tissue repair and responses . AlthougUSCs were first identified by via paracrine mechanisms, which can be activated by extracellular vesicles (EVs), including exosomes and microvesicles. Jiang et al. first extracted exosomes from USCs after finding spherical vesicles that were approximately 50–100 nm in size . USCs are adherent and usually begin to form clones on the 5–7th day, reaching confluence on the 10th day  protein (collagen type I) and 5% O2 hypoxia as an optimized approach allowing for the expansion of harvested USCs while maintaining their chromosomal stability and multilineage differentiation potential  with an hp-HA gel containing growth factors, and the cells exhibited an improved viability, engraftment ability and vascular, neural and myogenic differentiation potential after subcutaneous implantation in vivo  and progenitor cell culture medium  and observed that CD4+ T cell proliferation was inhibited in a dose-dependent manner and that the mRNA levels of IFN-γ, T-bet, IL-17A and RORC were significantly reduced. USCs can regulate CD4+ T cell activation and the Th1/Th17 immune response  significantly increases the USC expression levels of immunomodulatory cytokines (such as IL-6 and IL-8) and immunomodulatory chemokines (such as MCP-1). The secretion of cytokines  indicates that USCs have good immunomodulatory properties . Zhou etresponse . In addiresponse . Similarresponse . Additioresponse . HLA-DR response . All of Oxidative stress is a harmful process that can lead to a variety of diseases, including cancer, cardiovascular diseases, neurological disorders, respiratory diseases, kidney diseases, chronic inflammation and accelerated ageing . Upon stAngiogenesis is a critical process involving the repair and regeneration of tissues exposed to severe ischaemia/hypoxia  and is mediated by endothelial cell (EC) proliferation and migration . As the As an endothelial marker, CD31 has been widely used to evaluate the density of blood vessels. Zhao et al. discovered dramatically elevated CD31 levels in the islets of mice treated with low-dose streptozotocin (MLD-STZ) after USC transplantation, indicating that USCs can help rebuild the islet architecture . AdditioApoptosis is a programmed cell death process that involves the formation of distinct apoptotic bodies to maintain homeostasis and regulate the regular cell cycle. However, excessive apoptosis can further impair tissue function, which, in turn, leads to more severe pathological conditions . TherefoThe Bcl-2/Bax pathway is an important component of the endogenous apoptosis pathway. After the apoptosis pathway is activated, cells express the proapoptotic protein Bax to promote the release of cytochrome C (Cyt C) into the cytoplasm, which initiates the caspase cascade and leads to apoptosis. Sun et al. emphasized that USCs downregulated the expression of the proapoptotic proteins Bax and Caspase 3 and upregulated the expression of the antiapoptotic protein Bcl-2 in acute kidney injury (AKI) models. Compared with those in the control group, the renal function and histological damage of NRK-52E cells induced by cisplatin were significantly improved, and the proliferation of renal tubular epithelial cells was significantly increased . Similarvia posttranslational expression and significantly reduce NF-κB P65 nuclear localization, thus inhibiting the IRAK1-NF-κB signalling pathway  and increased expression of renal lineage-specific markers , suggesting that USCs have a significant renal cell-lineage differentiation ability. In addition, USCs can secrete growth factors such as VEGF and PDGF-BB and do not induce histological abnormalities, suggesting their safety nstrated .In vitro, the authors cocultured cisplatin-induced NRK-52E cells with USCs, and the cells exhibited a higher viability and lower apoptosis rate than the control group cells  , Lee et al. studied a partial cystectomy rat model and found a synergistic effect between USCs and heparin-immobilized bFGF-loaded scaffolds, as demonstrated by an elevated bladder capacity, compliance or decreased inflammation and tissue regeneration. These authors proposed that this novel composite biomaterial represents a promising therapeutic strategy for bladder reconstruction and provides the possibility for the structural and functional repair of damaged tissues; however, problems, such as vascularization and neurotization, remain and must be solved before clinical application . Li et ain vitro experiment did not necessarily reveal the same effect as the simulated experiment in the OAB models. Therefore, their study verifies only the effect of BK channels on USCs, and experimental data on the interactions of USCs are lacking. Whether USCs can serve as “vectors” to increase the effect of BK channels and therefore be useful as a treatment for OAB remains unknown.Overactive bladder (OAB), manifesting as urinary urgency and frequent nocturia, is often treated with invasive botulinum toxin or neuromodulation and reconstructive surgery . Large cInterestingly, the effect of USCs on underactive bladder (UAB) has been investigated. UAB is a complex urologic condition with severe complications, such as bladder outlet obstruction and stress urinary incontinence (SUI). Interstitial Cajal-like cells (ICC-LCs), pacemaker cells in the urinary system, have shown a downward trend in UAB  and haveUrinary incontinence (UI) is the involuntary leakage of urine through the urethra. SUI, the most common type of UI, is caused by the involuntary loss of urine caused by dysfunction of the urethral sphincter or the pelvic floor muscles. The prevalence of SUI ranges from 10 to 40% in females aged greater than 50 years .via vaginal balloon inflation, Wu et al. found that USC-Exos play a role in treating SUI, as demonstrated by improved urodynamic parameters, recovery of pubococcygeal muscle tissue, and the activation of the proliferation and differentiation of muscle satellite cells by the phosphorylation of extracellular-regulated protein kinases , which comprises both the vascular basement membrane and the interstitial ECM and serves as a microporous seeding-cell structure, and found that VECM is likely involved in angiogenesis based on its traits, which are similar to those of VEGF. Then, these authors differentiated USCs into urothelial and functional contractile smooth muscle phenotypes using induction media containing factors such as TGF-β1 and miR-199a-5p. Finally, these authors combined these cells with VECM to successfully construct an intact and multilayered urothelium and lamellar dense connective smooth muscle after 2 months of ureter reconstruction in a rat urethral defect model, while the control group exhibited only one layer of discontinuous epithelial cells and a few smooth muscle bundles. If the tensile and rupture strengths of such regenerated ureters are satisfactory, tissue-engineered grafts might be applied for the treatment of long-segment ureteral defects in the future . Liu et Diabetes is characterized by pancreatic β-cell destruction and insulin resistance and is well known as the ninth leading cause of death. Approximately 451 million people have been diagnosed with diabetes, and 5 million people died from its multiple comorbidities in 2018 or “and 5 million people have died from its multiple comorbidities since 2018 .” AlthouStudies have confirmed that isolated USCs can differentiate into insulin-producing β cells  and promvia a paracrine signalling mechanism, thereby improving left ventricular function and myocardial remodelling, rescuing glomerular function and increasing detrusor contractility , a cell-based therapy, can be used to treat congenital metabolic disease and acute and chronic liver failure in patients lacking suitable donors . Hu et ase model . In addise model . Moreovese model . All of Although studies investigating USCs in the digestive system are limited, we can reasonably infer the mechanism based on existing research. As mentioned above, USCs can participate in negative immune regulation. It is well known that the gastrointestinal tract is the largest immune organ in the human body and contains numerous immune cells, gut microbes and their metabolites, which can alter immune homeostasis. In contrast, alteration of the intestinal flora can upset this balance and lead to a series of inflammatory reactions . Thus, iDue to the absence of transplantable human neurons and insufficient technological means of neuron proliferation, challenges remain in the clinical treatment of neuronal apoptosis caused by spinal cord injury, acute brain ischaemia and neurodegenerative diseases . The capAfter seeding USCs onto hydrogel scaffolds and transplanting them into rat brains, Guan et al. found that USCs could not only survive at the lesion site with an excellent growth rate but also differentiate into neuron-like cells expressing neural phenotype-related proteins . In a stin vitro models, these authors found that USC-Exos could transfer microRNA-26a to induce the degradation of histone deacetylase 6 (HDAC6) mRNA and inhibit the translation of HDAC6 mRNA, thereby increasing the self-renewal and differentiation of NSCs  . Becausein vivo and can be used as a stem cell replacement for cartilage regeneration, providing a powerful platform for clinical transformation  and bone protein (OPG) proteins and thereby promote bone formation and inhibit bone formation. How these proteins are sorted into USC-EVs and then reused by recipient cells remains unclear, and their effectiveness in preventing osteoporosis requires further investigation . The abiormation . Some reSkeletal muscle is one of the largest organs in the human body. Healthy and complete skeletal muscle has decisive effects on human health and quality of life. Muscle injury and regeneration imbalance lead to the occurrence and development of diseases, and while muscle satellite stem cells (MuSCs) are the main source of muscle regeneration, their function gradually decreases over time. Therefore, alternative therapies are urgently needed . USCs hain vitro always leads to high cell death and low differentiation, researchers have explored novel methods to improve their survival rate in vivo by applying primary USCs to a hyaluronic-heparin hydrogel containing various GFs, such as myogenic-related IGF1, HGF, and PDGF-BB. The increased cell survival rate and the expression of myogenic and endothelial cell markers revealed the combinatory effect of USCs and GFs on muscle tissue engineering  in USC-conditioned medium, the authors observed significantly enhanced proliferation, motility and tube formation abilities of the HUVECs , faster reepithelialization and neovascularization, higher levels of collagen and higher capillary density . In an ie HUVECs . Bioglase HUVECs . By combe HUVECs . Scholare HUVECs . Moreovee HUVECs . USC-basHuman periodontal ligament stem cells (PDLSCs) are considered to be an important material for periodontal tissue regeneration and can be used in the treatment of diseases such as periodontitis and gingivitis. PDLSCs are largely not afflicted by the disadvantage of not allowing complete periodontal tissue regeneration affecting current clinical treatment strategies. However, due to the limited number of PDLSCs, developing strategies to promote cell proliferation and differentiation is currently the top priority.There is evidence of improved proliferation and osteoblastic/cementoblastic differentiation in PDLSCs, as demonstrated by denser collagen layers and elevated levels of the cementogenic protein cementum protein 1 (CEMP1), alkaline phosphatase (ALP) activity, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) in a noncontact coculture of USCs . AdditioThe average prevalence of ED, which is associated with many risk factors, such as diabetes, is 30% in patients of different ages. Due to their reduced quality of life, these patients often undergo high-cost penile prosthesis implantation, causing distressing complications, such as corporal fibrosis . Many stUSCs can improve erectile function and repair the endothelial structure of the cavernous body by protecting nerves, reducing fibrosis, and inhibiting cell apoptosis in cavernous tissue . In the Despite the substantial potential of USCs, there are still challenges that need to be overcome. When the cells used in experiments are derived from individuals with different genetic backgrounds, their diversity can affect the experimental reproducibility . The agein vitro, and their differentiation ability in vivo should be further explored. Preconditioning, including gene modification, drug pretreatment and coculture, can be used to enhance the differentiation ability of USCs in vivo (As mentioned above, USCs have multidirectional differentiation potential; however, given their decreased potential for osteogenic and chondrogenic differentiation compared with that of BMSCs, strategies to improve their directed differentiation efficiency are urgently needed . Moreove in vivo . Additio in vivo . However in vivo . These tin vitro amplification, administration and dosing, and cell homing must be optimized. In addition, the curative effect of basic cell therapy must be improved, and the cell therapy protocol must be optimized.Before USCs can be formally applied in clinical practice, standardized products must be acquired and storage and transport, Safety and effectiveness should also be considered. Recent research suggests that the proliferation and differentiation of implanted cells, such as USCs, and their corresponding scaffolds are in danger of reconstruction in the urinary tract due to the highly cytotoxic nature of urine .in vitro and even influencing disease modelling and clinical applications (Reprogrammed iPSCs from USCs are affected by genomic instability and epigenetic memory associated with the reprogramming process, resulting in aberrant differentiation ications . Teratomications .in vivo remains challenging (Exosomes derived from USCs represent a novel cell-free therapeutic strategy that have the advantages of long-term maintenance of biological activity, low immunogenicity and no promotion of tumour formation. Nevertheless, specifically labelling and tracking exosomes llenging .Overall, USCs and their secreted factors play roles in immune regulation, oxidative stress modulation, revascularization, cell apoptosis and autophagy. Through the mechanisms above, USCs have substantial potential in tissue engineering and personalized therapeutic strategies and exert therapeutic effects on a variety of diseases, such as those of the urinary, nervous, locomotor, reproductive endocrine and digestive systems. Although several challenges currently exist, we anticipate that future research will identify solutions to the existing problems and expand the application range.


Expectations regarding work , whether realized or not, may influence mental health, however there is limited quantitative research on this question. This study examined the longitudinal relationship between expectations of full-time work after age 62 and depressive symptoms and passive suicidal ideation among Baby Boomers, a generation that experienced the Great Recession as they neared retirement. Data came from the Health and Retirement Study, 2008 - 2016 . Clinically-relevant depressive symptoms were indexed by the Composite International Diagnostic Interview (CIDI). Expectation (probability) of working after age 62 was modeled continuously (range: 0 to 1). Multivariate mixed-effects logistic regression models of screening positive on the CIDI and passive suicide ideation were fit, separately, adjusting for demographics, household income and wealth, and health characteristics. Respondents working at baseline were less likely to screen positive on the CIDI longitudinally , and while expectations were inversely associated with screening positive on the CIDI this was not significant after accounting for work status . Longitudinally, higher expectations of working were inversely associated with passive suicidal ideation  even after accounting for working status. Future research will examine variation in these relationships by contextual factors like wealth, sex, and race/ethnicity to clarify how these features shape the association between work and mental health for this generation of older adults.


The role of the microbiota in human health and disease is well established, including its effects on several cancer types. However, the role of microbial dysbiosis in prostate cancer development, progression, and response to treatment is less well understood. This knowledge gap could perhaps be implicated in the lack of better risk stratification and prognostic tools that incorporate risk factors such as bacterial infections and inflammatory signatures. With over a decade’s research investigating associations between microbiome and prostate carcinogenesis, we are ever closer to finding the crucial biological link between the two. Yet, definitive answers remain elusive, calling for continued research into this field. In this review, we outline the three frequently used NGS based analysis methodologies that are used for microbiome profiling, thereby serving as a quick guide for future microbiome research. We next provide a detailed overview of the current knowledge of the role of the human microbiome in prostate cancer development, progression, and treatment response. Finally, we describe proposed mechanisms of host-microbe interactions that could lead to prostate cancer development, progression or treatment response. Whilege human ). Since arcinoma . In factarcinoma –12.Prostate cancer (PCa) is the second most frequent male malignancy worldwide with over 358,000 estimated deaths in 2018 . PCa hasThe advent of next generation sequencing (NGS) technologies has opened a new area of PCa research enabling unparalleled access to the genomic and transcriptomic underpinnings in PCa. Utilizing the potential for these technologies, several molecular markers have been proposed to stratify PCa , althougThe aim of this review is twofold, 1) provide the researcher with the necessary technical know-how to perform microbiome analysis, and 2) inform the reader of the advances that have been made in the field of prostate cancer microbiome research. We start out by describing in general how to analyse microbiome data and note several automated pipelines that are available to the researcher. For the sake of simplicity, we restricted our review to the three most frequently used NGS based analysis methodologies  that have been widely adopted by the microbiome research community. We next provide a detailed overview of the current knowledge of the role of the human microbiome in PCa development, progression and treatment response that was made possible by some of the aforementioned methodologies. Finally, we describe the proposed mechanisms of host-microbe interactions that could lead to PCa development, progression, or treatment resistance. A clear distinction between microbial association with PCa development (carcinogenesis) and its association with PCa progression (e.g. metastatic dissemination) is difficult to make due to the lack of healthy non-cancer control samples in most studies, and consequently this remains an outstanding question in the field.Most of the current research enumerating the microbial species present in the prostate and various other body sites utilize NGS based methodologies as opposed to the culture based techniques employed during the last century, which could detect only species that could be cultured. Three main methodologies are most commonly used now Fig. A. These https://github.com/najoshi/sabre) and quality checked  of the 16s DNA is amplified and sequenced. Next, for multiple samples sequenced in parallel, the raw sequencing reads are de-multiplexed (e.g. using sabre: g FastQC ) to remog FastQC ). If usig FastQC . Finallyg FastQC . These sg FastQC , mothur g FastQC , or qiimg FastQC . The datPhyloseq  is a powA limitation to amplicon sequencing is that only a particular gene region is amplified, which can bias diversity estimates depending on which variable region is selected. Further, different organisms can have different 16s gene copy numbers  which caUsing whole genome sequencing or total RNA sequencing as a way to detect non-host DNA/RNA has become a popular alternative to amplicon sequencing, as it enables species-level identification of organisms and generates a complete genome as well as a transcriptome for all the species, meaning that we can obtain information regarding the functional significance of the microbiome. Further, bacterial, viral, fungal and other archaeal reads can all be obtained using these methodologies, making them ideal for microbiome analyses. RNA sequencing based metatranscriptomic analysis can also shed light into which species are contributing actively to the expression profile of the tissue, whereas DNA sequencing based metagenome analysis captures all species, even though they might not be actively contributing to the tissue phenotype. Often, a combination of these two methodologies is required to understand the underlying tumor biology in the context of microbiome interactions with the tumor microenvironment.The bioinformatics workflow for metagenome or metatranscriptome analyses are similar to that for amplicon sequence based analyses, however with some key differences. Assuming that one has sequenced the entire microbial DNA/RNA pool from e.g. a human tissue sample, to an acceptable coverage, and has performed the pre-processing steps that are generic to raw sequence read analysis, including ribosomal RNA removal and paired-end read merging, then the first step is to align the reads to the reference human genome, e.g. hg38  using toMany automated bioinformatics pipelines such as Sunbeam , MetaWRAUsing these aforementioned methodologies, several studies have investigated the PCa associated microbiome as described in the following sections.Since 2015 there has been a steady rise in the number of publications looking at the association between the human microbiome and prostate cancer development, progression, and treatment outcome. While most research has focussed on the so called direct effect on PCa of the microbiome in the prostate tissue, others have also investigated associations between PCa and the core microbiota from different body sites, the so called indirect effects , 47, butTable With over a decade of research into understanding the role played by the prostatic microbiota in PCa pathophysiology, we are yet to find any causative organisms directly linked to prostate carcinogenesis, despite several studies indicating an association between certain species and the risk of PCa.We recently investigated the association between the prostate microbiome and PCa using a metatranscriptomic approach based on total RNA sequencing data from 94 PCa patients who underwent curatively intended radical prostatectomy for localized PCa . In order to investigate potential dysbiosis associated with PCa, we systematically compared the microbiomes between benign ) and malignant prostate tissue samples, between less vs. more-aggressive PCa, and between patients who suffered a biochemical disease recurrence compared to those who did not.Bacteroides fragilis, Saimiriine betaherpesvirus, Staphylococcus saprophyticus, and Vibrio parahaemolyticus had a significantly reduced abundance in the malignant as compared to the benign prostate tissue samples. Similarly, we observed a significant increase in the abundance of Shewanella in the malignant as compared to the benign prostate tissue samples, suggesting a perhaps important biological link between the prostate microbiota and PCa development. Within malignant tissue samples, those that had a higher abundance of Shewanella were associated with dysregulated host immune response, likely mediated by a decrease in enrichment of dendritic cells. We also observed a significant increase in the abundance of Microbacterium species in the T3 tumor samples as compared to the T2 samples, suggesting an association between advanced pathological stage and dysbiosis. While, the lack of true normal comparisons is a limitation to this study, obtaining tissue from healthy individuals is hard and ethically challenging.We revealed considerable dysbiosis associated with PCa. Notably, species such as Listeria monocytogenes, Methylobacterium radiotolerans JCM 2831, and Xanthomonas albilineans GPE PC73 to be negatively correlated with Gleason score, Tumor-Node-Metastasis (TNM) stage, and prostate-specific antigen (PSA) level, respectively. Microbes such as L. monocytogenes are known to play anti-tumor roles in PCa, for example by stimulating the innate and adaptive immune response [L. monocytogenes in the tumor as compared to AN samples, indicates a strategy wherein the microbes outcompete the tumor cells in the tumor microenvironment, enable recruitment of immune cells and thereby mitigate tumor growth.Other NGS-based studies have also correlated the abundance of microbial species with known risk factors for PCa. Using large scale whole transcriptome RNA sequencing data obtained from The Cancer Genome Atlas (TCGA) for 242 prostate adenocarcinoma (PRAD) patients from the United States and Germany, a recent study  found spresponse . The autNevskia ramose was found to have a positive correlation with Gleason score although its significance in PCa is not understood. Staphylococcus aureus was also found to be positively correlated with genomic alterations including amplifications in chromosome 19 and deletions in chromosome 15 and was associated with dysregulated immune-associated genes in this study [Furthermore, in this study , Nevskiais study , indicatis study , the higis study , preventEscherichia, Propionibacterium, Acinetobacter and Pseudomonas. Further investigation of their expression profiles revealed strong correlation between ten Pseudomonas genes and eight host small RNA genes. The authors noted that three of the host small RNA genes may be negatively associated with metastasis as they observed a high expression of these genes in a subset of patients with low rates of metastasis [Using both a metagenomic and a metatranscriptomic approach, a study investigating microbial dysbiosis in a Chinese cohort of 65 PCa patients  could notastasis . HoweverEscherichia and Acidovorax. This is perhaps not surprising considering the presence of geographic and ethnic variation in the composition of the human microbiome [In another metagenomic study comparing PCa microbiome of patients from different geographic/ethnic origins , significrobiome . The smacrobiome .Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes, fungal phyla such as Ascomycota, and Zygomycota, parasitic phyla such as Nematoda, and Sarcomastogophora, and group I and group IV viruses [A major stride towards establishing a microbiome signature for PCa was made using an array-based metagenomics and capture sequencing method . The stu viruses . Using h viruses .Of the indirect effects of the microbiome on prostate carcinogenesis, the association between gut microbiota and PCa has been studied the most. The human gastrointestinal tract harbors the majority of the bacterial population in humans surpassing that of any other bodily site by several orders of magnitude . CoevoluBacteriodes massiliensis as well as decreased relative abundances of Faecalibacterium prausnitzii and Eubacterium rectalie in the stool from men with PCa compared to the controls. A major limitation to this study [In a prospective case-control metagenomic study from 2018, stool samples from 20 Caucasian men with either benign prostatic conditions (n=8) or localized PCa (n=12) were analysed to evaluate their gut microbiome . The stuis study  is the sBacteroides and Streptococcus species in rectal swabs from patients with PCa (n=64) compared to non-cancer controls (n=41) were found in another study utilizing 16s rRNA amplicon sequencing [Bacteroides species was observed in patients with PCa compared to control men without PCa in this latter study [Similarly, enrichment of quencing . The cohquencing  utiliziner study , corroboer study , 59.Akkermansia muciniphila and Ruminococcaceae spp. were over-abundant in the fecal specimens of patients undergoing oral ATT compared to the other groups [While many studies have focused on the role of microbes in PCa development and progression, few have also investigated the changes to the microbiota following treatment against PCa. Using 16s rDNA amplicon sequencing, one such study  evaluater groups .Corynebacterium spp. in patients undergoing ADT, and an enrichment of Akkermansia muciniphila in patients taking oral AA, both compared to patients not receiving any form of treatment [Phascolarctobacterium and Ruminococcus as well as for several other bacterial species in patients who received ADT. Whether these insights could be exploited to enhance patient response to ADT needs to be investigated further.Similar findings were also observed in two other recent studies employing 16s rRNA amplicon sequencing methodology , 63. Usireatment . The secFew studies have investigated the association between the urinary microbiota and PCa. Urine samples are readily available, non-invasive, and have a higher patient compliance. Associations between the urinary microbiota and PCa can potentially serve as a biomarker that can be incorporated into pre-biopsy models to better predict PCa risk .E. coli and Enterococcus in the urine from PCa patients (n=13) compared to men (n=21) with benign prostatic hyperplasia (BPH) [Clostridium XVIII & IV, lachnospira, Acetanaerobacterium, and Faecalibacterium were found to be in significantly higher abundance in the urine from patients with PCa (n=14) compared to benign controls (n=16) as identified using 16s rDNA amplicon sequencing [Propionibacterium lymphophilum to have significantly higher abundance in PCa patients (n=61) vs. control men (n=63) who did not have PCa [One of the earliest studies evaluating the urinary microbiome and PCa using 16s rDNA PCR-denaturing gradient gel electrophoresis (DGGE) found significantly decreased abundance of ia (BPH)  in a Chiia (BPH) , speciesquencing . A priorhave PCa . Howeverhave PCa , 49, 60 Bacteroidetes, Alphaproteobacteria, Firmicutes, Lachnospiraceae, Propionicimonas, Sphingomonas, and Ochrobactrum, and a decreased abundance of Eubacterium and Defluviicoccus in the expressed prostatic secretions (EPS) from Chinese PCa patients (n=13) compared to Chinese men with BPH (n=21). In this study, qPCR detection of E.coli and Enterococcus revealed that E. coli was present in significantly higher number in the EPS and seminal fluid, whereas Enterococcus was present in significantly higher number in the seminal fluid of men with PCa compared to men with BPH [While it is interesting to profile the prostatic and seminal fluid microbiomes for associations with PCa due to their close proximity to the prostate gland, controlling for contamination from the urinary tract in these samples is often difficult. Few studies have evaluated the prostatic and seminal fluid microbiomes in PCa , 49, 64.Lactococcus, Carnobacterium, Streptococcus, Geobacillus, and Enterobacter, and decreased proportions of Cronobacter, Alkaliphilus, and Paenibacillus in prostatic fluid samples from patients with PCa (n=32) compared to men without PCa (n=27) in a Chinese cohort [Propionibacterium acnes genes were detected in the non-sperm fraction of the seminal fluid from Australian men with PCa (pooled sample from 6 men) but not in men without PCa (pooled sample from 6 men) using small RNA sequencing in a different study [Using 16s rDNA amplicon sequencing, another study found increased proportions of e cohort , whereasnt study . Most ofnt study , 64 are Cutibacterium acnes  is a skin-associated commensal that has been detected in the prostate of men with PCa in several studies [C. acnes has also been associated with chronic inflammation in the prostate of men with PCa [C. acnes isolates [C. acnes can induce cell proliferation [C. acnes infection [C. acnes responded via activation of transcription factors such as NF-κB and STAT3 [C. acnes-induced production of reactive oxygen species by keratinocytes in the skin, thereby inducing oxidative stress response and inflammation [C.acnes-induced inflammation in PCa development or progression.A number of studies have investigated the possible functional role of specific microbes in relation to PCa due to their inflammatory potential and frequent detection in prostatic tissue Table . Cutibac studies , 65 with studies , 67. C. with PCa  and showisolates . Evidencferation  and the feration , 71, whinfection . In anotnd STAT3 , which and STAT3 , 74. Othammation , an evenammation . Taken tE. coli in prostate carcinogenesis. Uro-pathogenic strains of E. coli are known to induce prostate tissue damage in rat models of prostatitis [E. coli for 12 weeks developed chronic inflammation in the prostate, and with prolonged infection showed cytological changes typical for prostatic intraepithelial neoplasia and high-grade dysplasia. Increased epithelial cell proliferation, and oxidative DNA damage was observed in the prostate glands exhibiting dysplasia, together with decreased androgen receptor and PTEN gene expression, as compared to the control glands [E. coli-induced inflammation and the onset of PCa or pre-neoplastic lesions.Many studies have also investigated the role of statitis , mediatestatitis . In a mostatitis , all micl glands . This coFaecalibacterium prausnitzii have also been associated with PCa, with higher fecal abundances of F. prausnitzii observed in benign as compared to malignant patient samples [F. prausnitzii is generally considered to have anti-inflammatory properties with its ability to produce butyrate and induce secretion of anti-inflammatory cytokines such as IL-10, TGF-β2 and IL-1Ra . Furtheell line  and coulell line .Ruminococcus gnavus and Bacteroides acidifaciens were enriched in the fecal microbiota of castrated (as compared to non-castrated) male mice, and that these species can metabolise androgen precursors, pregnenolone and hydroxypregnenolone, into downstream metabolites of the androgen biosynthesis pathway, dehydroepiandrosterone and testosterone. As prostate tumors are reliant on androgens for continued growth, such an alternate source of androgen could lead to endocrine resistance in PCa patients undergoing castration treatment as shown in PCa mouse models [While mechanistic studies investigating microbial regulation of host metabolism in men with PCa are limited, a recent study  using PCe models .V. parahaemolyticus indicating altered host immunity associated with the presence of V. parahaemolyticus . Moreover, malignant prostate tissue samples having high (vs. low) abundance of Shewanella showed decreased enrichment of dendritic cells and down-regulation of several toll-like receptors crucial for an active immune system , perhaps enabling tumor-immune evasion. Similarly, another recent study [Delftia acidovorans, Gardnerella vaginalis) abundance in PCa tissue and regulatory T-cells, as well as with down-regulated immune-associated genes including LPCAT2, TL3, and TGFB2, indicating an immuno-suppressive tumor environment associated with the PCa microbiota.Disentangling host immune responses against tumor cells from those induced by microorganisms is often difficult. We have recently shown enrichment of several immune cell types within malignant prostate tissue samples having low vs. high abundances of nt study  found stRuminococcus could be a top candidate for further investigations of its possible driver role in development of treatment resistant CRPC. Conceivably, future prospective clinical trials could test if a novel treatment strategy aimed at eliminating Ruminococcus sp. from the gut of men with PCa, is able to prevent/delay treatment resistance and hence improve survival. Future research should also combine metagenomic or metatranscriptomic investigations with metabolomics to provide a more complete overview of host-microbe interactions within the context of PCa. Research should also focus on delineating novel blood and/or urine based microbial signatures that could be of diagnostic, prognostic and/or predictive potential.Future research should focus on mapping host microbial species to their functional profiles and delineating specific mechanisms by which microbes enable cancer development and progression or affect treatment response. A better understanding of host-microbiome cross-talk and the associated molecular mechanisms could pave the way for development of novel prevention and/or treatment strategies. Thus, future studies should investigate the clinical utility of targeting the microbiome as a novel mode of anti-cancer treatment. As an example, the genera Through this review we aimed to explore the extent of microbial dysbiosis that is associated with PCa by providing an overview of the current knowledge in the field. Moving away from the notion of cancer as solely being a disease of the genome, we believe a more holistic approach towards cancer treatment, informed by genetic, epigenetic, and host-microbiome interactions could benefit treatment decisions in the future. PCa, like other cancers, is a dynamic and heterogeneous disease that has several layers of molecular and cellular complexity associated with it. Microbiome analyses have revealed a bacteria rich environment in the prostate that might be altered during disease onset, progression or treatment. Several species of the microbial community have been associated with PCa aggressiveness and response to therapy, a finding that has also been observed in many other cancer types. A better understanding of the role microbes play in these processes will help us develop novel treatment strategies as well as better risk stratification tools. For instance, it is possible that removal of certain gut microbial species prior to androgen deprivation/hormonal therapy could delay disease progression to CRPC. Several clinical trials are underway looking at e.g. the efficacy of fecal microbiome transplant in combination with established treatment strategies as a means to control tumor progression in different cancers, including in PCa. If successful, these could give us an upper hand in the battle against cancer.


Overall, CRISPRa represents a valuable addition to the experimental toolbox for neuronal cell-type programming.Gene activation with the CRISPR-Cas system has great implications in studying gene function, controlling cellular behavior, and modulating disease progression. In this review, we survey recent studies on targeted gene activation and multiplexed screening for inducing neuronal differentiation using CRISPR-Cas transcriptional activation (CRISPRa) and open reading frame (ORF) expression. Critical technical parameters of CRISPRa and ORF-based strategies for neuronal programming are presented and discussed. In addition, recent progress on  Induced expression of desired genes has been an important strategy for revealing gene function and for modulating cellular activity for synthetic biology and therapeutic applications. Apart from ectopically expressing additional copies of a gene by introducing their open reading frames (ORFs), methods to artificially activate endogenous copies of genes have been explored, including transcription activating factors tethered to zinc finger proteins  and tranin vitro and in vivo applications of CRISPRa with a particular emphasis on neuronal cell fate engineering in comparison to ORF expression strategies.To achieve gene activation with CRISPR-Cas9, a catalytically deactivated Cas9 (dCas9) was created to bind to specific genomic regions without the ability to create a double-strand break . To endoin vitro engineering of cell types that are present in the human body has many important applications, including studying cellular functions, investigation of disease progression, development of therapeutic interventions, and cell-based transplantation therapies. Being recognized for their key roles in modulating gene expression, TFs have been broadly used for cell fate engineering, most of which utilized cDNA or ORF overexpression. Pioneering work in 1987 showed that cell identity could be altered by forced expression of a single TF MyoD, which converted fibroblasts into myoblasts  . These dOne potential advantage of the CRISPRa system relates to epigenetic modifications around the endogenous loci, which are hypothesized to better mimic natural epigenetic marks. Evidence for this remodeling process was investigated by delivering gRNAs targeting promoter regions of Ascl1, Brn2 and Myt1l to convert mouse embryonic fibroblasts (MEFs) into neurons . A dCas9The CRISPR-Cas system is easily scalable to highly multiplexed screens using complex gRNA libraries because of the short length of gRNAs compared to full-length TF ORFs . In thisCRISPRa screening for neuronal differentiation has also been carried out in hiPSCs by using a library of 8,435 gRNAs targeting 1,496 TFs in the human genome. A dCas9 with VP64 fused to both N and C termini was used for gene activation and a TUBB3-2A-mCherry reporter was generated for indicating neuronal cell fate . After cin vivo neurons, a 75.5% overlap of enriched genes was observed when comparing iNs or in vivo neurons to MEFs. However, it was unclear whether the magnitude of enrichment (i.e. expression fold-change and/or significance) was similar, but these results do highlight the convergence to a “core neuronal transcriptome” when diverse TF pairs are used. Because endogenous neurons have many subtypes, the heterogeneity amongst iNs created from different TF pairs was investigated to see if different TF pairs could produce different subtypes of neurons. Groups of iNs that expressed higher levels of nicotinic receptor genes were identified, also exhibiting preferred responses to nicotine stimulation in calcium imaging experiments. Overall, only a limited number of genes provided a confident similarity of resulting iN cells to endogenous subtypes. This observation indicates that even though different pairs of TFs could produce different gene expression profiles on top of the core neuronal programs, they are not yet specific enough to produce the vast diversity of neurons that exist in the mammalian brain. Combinatorial and serial screening of TFs for recreating neuron subtypes in vitro is still in its early stages. Although different pairs of TFs can reprogram fibroblasts into induced neurons, it is important to note that even the same pair of factors can reprogram other non-neuronal cells into different types of neurons: a study  . TF pair a study  showed t a study .Combining single-cell transcriptomic readout with pooled TF activation in stem cells is a high-throughput way to establish causality between TFs and their effects on cell fate. Along this line, a proof of concept screen with 61 barcoded ORFs demonstrated the feasibility of associating TF expression with specific single-cell transcriptome clusters . Clusterversus CRISPRa is the ability to elevate expression of a certain TF isoform. When the four isoforms of ETV2 were compared for inducing endothelial cell differentiation, only isoform 2 was able to generate cells that have near complete VE-cadherin expression and possess high tubulogenic capacity. ETV2-programmed cells are useful for engineering vascularized brain organoids  . It is cGene isoforms generated from alternative splicing events and/or TSSs are prevalent in the human transcriptome , and plaGene-gene interactions are also important for cell differentiation and function. Because of the short gRNA length, CRISPR systems could be more amenable to combinatorial screening. This is enabled by several factors: the ability to synthesize specific combinations of gRNAs allows for a reduction of the vast combinatorial search space, and the capacity to readily package more gRNAs in tandem into viruses or other vectors compared to ORFs. Despite this notion, combinatorial genetic screening using CRISPRa has yet to be demonstrated convincingly, as only individual genes and select pairs of genes have been screened using CRISPRa. Deep combinatorial screens of many combinations of genes remain unexplored using CRISPRa. One limitation arises from the need for more orthogonal promoters to express gRNAs in order to avoid recombination of the same promoter. It was reported that lentiviral gRNA expression vectors with identical U6 promoters were prone to recombination and loss of gRNAs . Thus, pvia the activation of gene regulatory networks. This is important so that reprogrammed cells can maintain their identity without the need for continuous differentiation signals and is one of the marks of truly programmed cell identity. Theoretically, CRISPRa systems are better than ORFs at activating endogenous loci, because gRNAs are targeting the genome directly, while ORFs would need a positive feedback loop to induce expression of the endogenous loci. However, this is only partially supported by a transient transfection experiment that observed higher levels of endogenous transcripts and chromatin modifications in CRISPRa transfected cells than the ORF groups  and gRNA cassettes, while ORF strategies need co-delivery of multiple ORFs. The best approach to achieve specific goals should be evaluated on a case-by-case basis. For gramming  and ninegramming . For a lgramming , pooled tivators  and immuin vitro and in vivo neuronal programming. We also highlighted multiplexed neuronal differentiation screens using CRISPRa or ORF expression. Critical technical parameters of these strategies to consider are gene expression levels, gRNA specificity and strength, gene isoform control, scalability for combinatorial testing, stable cellular programming, as well as gene and gRNA delivery. To harness the full potential of CRISPRa for combinatorial screening approaches, careful and rational design of gRNA libraries, scalable readout of perturbation/effects, and statistically robust analysis pipelines are needed. As demonstrated in aforementioned studies, there is a tendency to validate gRNA hits with ORF overexpression as a gold standard in part because of potential off-target effects from gRNA/CRISPR. There are many examples showing that ORF strategies are better suited for generating stable inducible cell lines required for downstream characterizations and applications of the programmed neurons.Here, we summarized recent developments using CRISPRa for in vitro cell fate studies (in vitro derived brain organoids as part of different brain areas, only by measuring the gene expression profile (As a readout, single cell RNA-seq allows cell type heterogeneity of multiple organs to be resolved at an unprecedented level . Single  studies . Due to  studies , researc studies , as succ studies . In line studies , it is p profile . In forw


Hodgkin lymphoma (HL) is a highly curable disease; proton therapy for mediastinal HL irradiation might theoretically reduce late toxicities compared with classical radiotherapy techniques. However, optimal patient selection for this technique is subject to debate. While implementation at a larger scale of proton therapy for HL may face organizational, political, and societal challenges, new highly effective systematic drugs are being widely evaluated for this disease.Consolidative radiation therapy for early-stage Hodgkin lymphoma (HL) improves progression-free survival. Unfortunately, first-generation techniques, relying on large irradiation fields, were associated with an increased risk of secondary cancers, and of cardiac and lung toxicity. Fortunately, the use of smaller target volumes combined with technological advances in treatment techniques currently allows efficient organs-at-risk sparing without altering tumoral control. Recently, proton therapy has been evaluated for mediastinal HL treatment due to its potential to significantly reduce the dose to organs-at-risk, such as cardiac substructures. This is expected to limit late radiation-induced toxicity and possibly, second-neoplasm risk, compared with last-generation intensity-modulated radiation therapy. However, the democratization of this new technique faces multiple issues. Determination of which patient may benefit the most from proton therapy is subject to intense debate. The development of new effective systemic chemotherapy and organizational, societal, and political considerations might represent impediments to the larger-scale implementation of HL proton therapy. Based on the current literature, this critical review aims to discuss current challenges and controversies that may impede the larger-scale implementation of mediastinal HL proton therapy. Hodgkin lymphoma (HL) is characterized by a high curative rate, evaluated between 80% and 90% . ConsoliMultiple strategies have been proposed to select patients for proton therapy. These approaches can be based on dosimetric parameters, normal tissue complication probability (NTCP) models, or cost-effectiveness considerations .In the context of limited resources, a selection process should logically take place to reserve proton therapy for the HL patients who would benefit the most from this technique. The ILROG guideline consensus about proton therapy for HL patients  currentlCurrent ILROG guidelines for HL radiotherapy  recommenBased on the dosimetric comparison between proton therapy and optimally planned IMRT, dose to OAR and planned target volumes coverture can be compared. A patient selection can be made at this level based on the absolute dosimetric gain.Based on a retrospective analysis of HL patients treated with proton therapy, dosimetric selection criteria could be proposed . Such crBased on a cohort of 21 patients treated with DIBH IMPT, Ntentas et al.  noted thThis selection method is simple to implement but presents some limitations. A dosimetric gain is not necessarily informative on the potential clinical benefit for the patient, as evidenced by non-significant or low toxicity gain when applying NTCP models as discussed in the first section ,19. The Dosimetric superiority does not necessarily translate into clinical benefit; consequently, NTCP models have been used to predict expected radiation-induced toxicity reduction to ultimately select patients who may benefit the most from proton therapy. Langendjik et al.  proposedScorsetti et al.  proposedWhen a patient can develop multiple late toxicities and when the clinical decision is based on a composite score, the question of differentially weighting toxicities should be considered: all OAR late toxicities do not have the same functional impact or prognosis for the patient. Carbini et al.  proposedWhile the NTCP model may probably be more clinically relevant than crude dosimetric parameters, some pitfalls associated with NTCP calculation must be kept in mind.First and foremost, NTCP models have been retrospectively developed with a given radiotherapy technique, at a precise moment. Troeller et al.  unambiguIn addition, the proposition of the selection threshold is subjective and consequently debatable. Using three different selection rules based on unweighted toxicity scores, Scorsetti et al.  demonstrFurther approaches have been proposed to weigh late radiation-induced toxicities by taking into account the related life-year-lost (LYL) estimate. Rechner et al.  comparedTo take account of the financial cost of HL proton therapy compared with standard radiotherapy, some cost-effectiveness models are currently being developed for HL patients. Vega ,34 evaluTo increase the cost-effectiveness of HL proton therapy, systemic improvements must be made. Bortfeld et al.  considerThe financial sustainability of particle radiotherapy is challenged by the commissioning of effective photon therapy techniques which might lead to high WTP per QALY to be cost-effective for proton therapy. A recent dosimetric study by Moreno et al.  evaluateTo ease HL proton therapy treatment the necessity of DIBH has been questioned by some authors. Everett et al.  demonstrThere is currently a global worldwide trend to reduce the dose and the consolidative radiotherapy indications in HL management, which might be motivated by fear of late adverse events and the development of new effective drugs that might challenge radiotherapy. The HD10 trial  demonstrBrentuximab Vedotin (BV) has been recently evaluated in the frontline context. Kumar et al.  found thHL proton therapy faces worldwide reimbursement issues, which heavily depend on the country’s insurance system. To exemplify this diversity, the Dutch health system currently reimburses HL proton therapy when an expected clinical benefit is demonstrated based on NTCP models . The FreThe inequality of access at a national and international level represents an additional challenge for larger-scale HL proton therapy implementation. Belard et al.  tested tFinally, at the proton therapy center level, the capacity of treatment might be limited by the number of the treatment room. With limited disponibility, HL might not be prioritized over other indications for proton therapy for which the level of evidence of particle therapy is higher  and over patients with in-place tumors ,61.The development of proton therapy for HL is currently facing multiple challenges. Optimal patient selection for HL proton therapy is complex and subject to intense debate. In addition, one should acknowledge the accelerating development of new effective systemic treatments for HL disease. Furthermore, technical, organizational, and societal questions will have to be tackled to implement at a larger scale proton therapy for mediastinal HL.Nevertheless, proton therapy should theoretically reduce secondary malignancies and late radiation-induced toxicities thanks to its minimal distant-to-target dose deposition. While selection criteria are yet to be precisely defined, specific patient populations may readily benefit from proton therapy such as young female patients with the lower mediastinal disease.