Nucleic acid amphiphiles, referring to nucleic acids modified with large hydrophobic groups, have been widely used in programmable bioengineering. Since nucleic acids are intrinsically hydrophilic, the hydrophobic groups endow nucleic acid amphiphiles with unique properties, such as self-assembling, interactions with artificial or biological membranes, and transmembrane transport. Importantly, the hybridization or target binding capability of oligonucleotide itself supplies nucleic acid amphiphiles with excellent programmability. As a result, this type of molecule has attracted considerable attention in academic studies and has enormous potential for further applications. For a comprehensive understanding of nucleic acid amphiphiles, we review the reported research on nucleic acid amphiphiles from their molecular design to final applications, in which we summarize the synthetic strategies for nucleic acid amphiphiles and draw much attention to their unique properties in different contexts. Finally, a summary of the applications of nucleic acid amphiphiles in drug development, bioengineering, and bioanalysis are critically discussed.  Ma and colleagues review the development on nucleic acid amphiphiles referring to nucleic acids modified with large hydrophobic groups. They summarize the synthetic strategies, highlight the unique properties , and delve deeper into the applications . The biological functions of nucleic acids include carrying genetic information (genomic DNA and mRNA), regulating gene expression , and biocatalysis (ribozymes). With the development of nucleic acid synthesis methods, nucleic acid-based nanotechnology, therapies and diagnostic tools have accomplished impressive achievements over the last few years.Nucleic acid amphiphiles with increased hydrophobicity have high affinity toward the cellular membrane and unique self-assembly properties. Thus, nucleic acid amphiphiles have proven to be an effective strategy for nucleic acid drug delivery and building blocks for constructing nanostructures that have applications in biological contexts. In this review, we first thoroughly discuss the synthesis methods of nucleic acid amphiphiles by comparing the pros and cons of solid-phase and liquid-phase coupling methods and review recent progress in synthesis method development. Next, the unique properties of nucleic acid amphiphiles, including self-assembly and interactions with membranes are discussed in more detail. Finally, the applications of nucleic acid amphiphiles in drug delivery, cell membrane engineering, and bioanalysis are critically discussed for diagnosis and treatment.Nucleic acids are usually synthesized using an automated DNA/RNA synthesizer with phosphoramidite chemistry on solid supports (controlled pore glasses). The phosphoramidites derived from protected nucleosides are coupled through a four-step reaction cycle to assemble the nucleic acid chain. After chain assembly, the nucleic acid chains are cleaved from the solid supports and deprotected to obtain the final product. During this process, various hydrophobic moieties such as lipidic polymers, endogenous lipids, large hydrophobic groups, and DNA intercalators can be attached to the nucleic acids, resulting in nucleic acid amphiphiles . The synBased on the conjugation position of hydrophobic moieties on nucleic acids, nucleic acid amphiphiles can be classified into three categories: 5′ terminal-functionalized, 3′ terminal-functionalized, and internal-functionalized nucleic acid amphiphiles. The phosphoramidite chemistry-based solid-phase synthesis method is commonly used for nucleic acid amphiphiles, which enables the synthesis of all oligonucleotide/oligodeoxynucleotide variants . However, the synthesis strategies vary depending on the conjugated hydrophobic moieties. In this review, we discuss the primary hydrophobic modifications utilized in solid-phase synthesis.,,,,,,,Because the synthetic direction of the solid-phase DNA/RNA synthesis is from the 3′ terminal to the 5′ terminal, 5′ terminal modification of hydrophobic groups is easily accomplished through a standard DNA/RNA synthesis procedure using hydrophobic group phosphoramidites A. To datin vivo and cell delivery efficiency. Although conjugating a nucleic acid drug with a hydrophobic group can improve the delivery efficiency of nucleic acid drugs, it is crucial to determine the conjugated locations of hydrophobic groups. For instance, 3′ terminal modifications of siRNAs significantly enhance the resistance to 3′ exonucleases, which are highly expressed in serum and intracellular conditions.,Although it is easier to conjugate hydrophobic moieties at the 5′ terminal of nucleic acids, conjugation to the 3′ terminus is also necessary in circumstances. For nucleic acid drug development, two significant challenges are circulatory stability ,,,,Internal-functionalized nucleic acid amphiphiles can be obtained by incorporating hydrophobic group-modified phosphoramidites. Hydrophobic moieties can be modified on nucleotides either at the 2′ position of the sugar ringThe solid-phase modification method for nucleic acid amphiphile synthesis is advantageous for its convenience, high efficacy, and high yield. However, the use of robust deprotection conditions  hinders its utility in synthesizing ester/halogen-based nucleic acid amphiphiles. In addition, limited scalability in synthesis  and high reagent consumption further constrain its application.,,22), stearic acid (C18), palmitic acid (C16), a branched (bis-C10) N,N′-didecyl chain, chromophore pyrene, and preformed polystyrene could condense in the micellar core owing to hydrophobic interactions. An increased local concentration of reactive groups in the micellar core significantly improved the conjugation yields . Based on theoretical derivations, if we assume that chalcone-dihexadecane is equivalent to a cholesterol molecule plus two C12H22) to the 5 position of uridine. Then, incorporation of dodecane moieties with varied positions and quantities was allowed by a DNA/RNA synthesizer. As expected, when they increased the amount of modified lipidic dodecanes, the CMC of the resulting conjugates decreased.Similarly, Liu et al. reported that nucleic acid amphiphiles conjugated with diacyllipid have a CMC lower than 10 nM, whereas those conjugated with less hydrophobic monoacyllipid cannot form micelles even at millimolar concentrations.,,,,,4, PEG8, and PEG24), alkyl (C12 and C24), or oligonucleotide  have higher binding affinity than NoSPR but do not restore affinity to that of the free aptamer.The ability to hybridize with complementary strands or recognize target proteins is another parameter for further application of nucleic acid-formed micelles. In most cases, the hybridization capability of nucleic acids is preserved.,,,,+) base pairing in acidic conditions which dissociates into random coils in slightly basic conditions,in situ transition between diblock and triblock structures through dissociation and formation of i-motif structures upon pH changes. They established a method of morphology-shifting from spherical micelles to nanofibers , the hydrophobic/hydrophilic ratio of the nucleic acid amphiphiles appears to play a vital role in determining the morphology of the formed nanostructures.nofibers C.53 Likeoperties E.55 Such,,,,Liposomes are commonly used as models to study interactions between nucleic acid amphiphiles and membranes. Moreover, functionalization of liposomes with nucleic acid amphiphiles has been proven to be a convenient method of constructing liposome-based drug delivery systems and biosensors.,2+, and temperature.,Because nucleic acid-mediated liposome assembly has certain application potentials in the field of nucleic acid detection, Vogel’s group conducted research on nucleic acid-mediated liposome assembly in this field’s early days.,,,,Nucleic acid-mediated particle assembly is a topic that has been studied for a long time. Similar research on inorganic nanoparticle assembly was conducted considerably earlier and is more established than that of nucleic acid-mediated liposome assembly. Research on nucleic acid-mediated liposome fusions is more representative of the uniqueness of nucleic acid amphiphile-modified liposomes. Membrane fusion occurs in various important biological processes, such as cell endocytosis, exocytosis, and virus infection. This process generally consists of three steps: (1) the membranes approach each other, (2) the original membranes undergo deformation and rearrangement, and (3), the contents of the membranes mix to exchange their inner material.To summarize, nucleic acid amphiphile-induced liposome assembly and fusion have been thoroughly studied over the past two decades and considerable knowledge has been gained. Great achievements have been witnessed on the application of these models, such as microRNA detection by DNA-mediated liposome fusionUnderstanding the interactions between nucleic acid amphiphiles and the cell membrane would be useful for their applications in cell membrane engineering and nucleic acid drugs delivery.,,,in vivo or in serumFirst, whether the nucleic acid amphiphiles can be stably anchored on the cell membrane is closely related to the hydrophobic group. In general, nucleic acid amphiphiles gain a higher insertion efficiency with greater hydrophobicity. Hydrophobicity can be regulated by different types and number of hydrophobic groups,,Linkers between nucleic acids and hydrophobic groups also have an effect on amphiphiles anchoring on cell membrane.,The length of the nucleic acid chain also has a profound impact on the membrane insertion efficiency of nucleic acid amphiphiles. The insertion efficiency of nucleic acid amphiphiles into lipid membranes decreased with longer nucleic acid segments. This can be attributed to the increased hydrodynamic pull of the fully hydrated negatively charged backbone, which increases steadily with length extension. Liu et al. held the opinion that nucleic acid amphiphiles with long nucleic acid chains form micelles with greater charge density,After the nucleic acid amphiphile is anchored to the cell membrane, it may be endocytosed into the cells through the endocytosis pathway.As expected, the structures of nucleic acid amphiphiles play a vital role in determining their properties. When designing a nucleic acid amphiphile for a specific application, its properties are predicted through rational design based on the knowledge gained from previous basic research. At the same time, experimental data can help advance our understandings of the refined framework of the structure-properties relationship for nucleic acid amphiphiles.The research discussed above on the synthesis and properties of nucleic acid amphiphiles lay the foundation for their further biological applications. Self-assembly of nucleic acid amphiphiles, nucleic acid amphiphile-modified liposomes, and single nucleic acid amphiphiles have found applications in drug delivery. Nucleic acid amphiphiles and nanostructures formed from nucleic acid amphiphiles with the ability to anchor stably onto cell membranes have been applied in cell membrane engineering and analysis. Recently, the mechanism of nucleic acid amphiphile-induced liposome fusion was applied for microRNA detection. These applications are discussed in detail in this section.trans-4-hydroxyproinol linker,,,,,,d ∼1 μM), which extends the serum half-life of siRNA from <10 to ∼90 min.,,,,,Coupling highly hydrophobic molecules to nucleic acid drugs may improve delivery efficiency into cells. Taking hydrophobic siRNA as an example, chol-siRNA, known as the famous “cholesterol conjugated siRNA,” has been studied for decades. Terminal modification of cholesterol through the ,,Apart from the delivery of nucleic acid drugs, the micelles formed by nucleic acid amphiphiles are also used in the delivery of poorly water-soluble drugs B.144,145,,,,In addition to improving delivery efficiency, conjugating nucleic acid drugs with highly hydrophobic groups might also improve the activity of the modified nucleic acid drug. Researchers such as Xodo and co-workers have explored the impact of polycyclic aromatic hydrocarbon units  on the folding, stability, and potency of triplex-forming oligonucleotidesCollectively, nucleic acid amphiphiles have the potential to improve the delivery efficiency of nucleic acid drugs and small-molecule drugs. In addition, hydrophobic moieties can participate in the structure formation and target recognition of nucleic acid amphiphiles. In addition to being used in nucleic acid delivery systems, nucleic acid amphiphiles have been used for small-molecule delivery or enhancing the activity of nucleic acid drugs, though its application is still in infancy.Under specific conditions, nucleic acid amphiphiles can anchor to cell membranes. Benefiting from diversity and programmability of nucleic acid structures, many applications related to cell membranes have become possible, including modeling cell-to-cell connections, simulating the function of transmembrane proteins using nucleic acid nanostructures, and analyzing biological events near the cell membranes. Compared with covalent modification of cell membranes and functionalizing cell membranes through genetic engineering, anchoring of nucleic acid amphiphiles to cell membranes is a simpler and harmless method. Such cell membrane engineering approaches cannot only endow cells with new functions and make new cell therapies possible but also deepen people’s understanding of related biological issues through a bottom-up process.,,An important research area of cell engineering is the use of nucleic acid amphiphiles (mainly composed of nanostructures conjugated with lipidic molecules) to simulate the functions of membrane proteins. The nucleic acid nanopores can be used to simulate the transport channels onto the cell membrane. Typically, these transmembrane channels consist of membrane proteins, which facilitate the transport of water, ions, and other entities. The significance of simulating the transmembrane channel functions with nucleic acid nanostructures is that the size and shape of the nanopores can be tuned more conveniently to meet specific requirement.16 alkane-nucleic acids had the highest incorporation efficiency.Another important application in cell membrane engineering is the rebuilding of intercellular interactions. For nucleic acid amphiphiles, either the hybridization properties of nucleic acids or specific structures such as aptamers are utilized to simulate a series of biological processes such as interactions between cells and adhesion of cells to matrix. The rebuilding of intercellular interactions is the foundation of tissue rebuilding.2+ and K+ near the cell membrane. Gas molecules are also a common type of signaling molecules. However, so far, gas-responsive nucleic acid molecules have not been reported. Interestingly, Feng et al. constructed a small molecule that could react with HSO3− through Michael addition. This molecule produces strong fluorescence upon binding to the G-quadruplex, and the fluorescence intensity decreases after reacting with HSO3−. Thus, the researchers obtained a nucleic acid-based SO2 biosensor. Similarly, they constructed another G-quadruplex-based NO biosensor using the same design principle. These cell membrane-anchored gas molecule responses enable real-time monitoring of gas signal transmission.Cell membrane plays vital roles in biochemical events. The detection of microenvironmental changes near/on the cell membrane is of great significance to the understanding of related biochemical events. Nucleic acid amphiphiles that can be anchored to the cell membrane, with a combination of specific anchoring properties of the lipid part and the high functional diversity of the nucleic acids, have been developed for the detection of a variety of signal substances in the cell membrane microenvironment , such asThe extracellular pH is related to a variety of physiological and pathological processes, hence the detection of extracellular pH has always been a research hotspot. Ke et al. firstly tried to apply nucleic acid amphiphiles to construct ratiometric fluorescent probes for the detection of extracellular pH. In their study, the hydrophilic nucleic acids of amphiphiles were merely regarded as a linker between the lipidic part that anchored in the cell membrane and the pH-responsive fluorescent molecules, which guaranteed that the pH-responsive moieties would protrude from the cell.In addition to detecting changes in the microenvironment near the cell membrane, nucleic acid amphiphiles are also used to study the cell membrane itself.During the past two decades, nucleic acid amphiphiles have been successfully applied to construct various nanostructures and have achieved a variety of applications in biomedicine and bioanalysis. In this paper, we review the solid-phase and solution-phase synthetic strategies that have been reported for the synthesis of nucleic acid amphiphiles. We then discuss the properties of nucleic acid amphiphiles in various contexts, including self-assembly properties, and interactions with liposomes and cell membranes. Finally, we explore the applications of nucleic acid amphiphiles in drug development, cell membrane engineering, and bioanalysis. Next, we propose several directions for this research area. Despite the diverse nucleic acid amphiphiles utilized to date, a reliable large-scale synthesis method for biomedical applications is still lacking. Solution-phase synthesis is generally considered more suitable for preparing nucleic acid amphiphiles on a larger scale. However, due to the difference in molecular polarity between nucleic acids and hydrophobic ingredient, it is quite challenging to remove the excessive hydrophobic ingredients completely, which often encase the target nucleic acid amphiphiles during the concentration process and decreased final yield of nucleic acid amphiphiles. Therefore, it is essential to explore novel synthetic strategies for large scales, which would benefit the transformation of nucleic acid amphiphiles from being limited to basic research to practical application. To study the properties of nucleic acid amphiphiles, it is critical for researchers to bridge the gap between the research and specific applications. Taking the relationship between nucleic acid amphiphiles and membranes as an example, the considerations vary with different applications. For membrane protein simulation, researchers pay more attention to the anchoring stability of the nanostructures formed by nucleic acid amphiphiles, while for drug delivery, researchers pay more attention to the transmembrane transport of nucleic acid amphiphiles. Thus, the structure-property relationship gained from research could guide the rational design of nucleic acid amphiphiles for specific applications. Furthermore, the properties of nucleic acid amphiphile observed during practice for specific applications would further expand our understanding of the rules behind nucleic acid amphiphile self-assembly and interactions with membranes. Currently, there is no standardized method to predict the properties of a nucleic acid amphiphile with a specific structure. More research with varied nucleic acids, hydrophobic moieties, and linkers is still needed to expand our understandings. After sufficient data have been collected, it may be possible to predict the properties of newly emerged nucleic acid amphiphiles using machine learning methods in the near future.Finally, most research on nucleic acid amphiphiles focus on self-assembly, anchoring to lipid surfaces, and oligonucleotide delivery. However, the interactions between nucleic acid amphiphiles and biomacromolecules are rarely reported, except in several of the cases reported in this review, which deserve further study. Anyway, it is foreseeable that, as our understanding of nucleic acid amphiphile properties deepens in the future, the application scope of nucleic acid amphiphiles will greatly expand.


The SARS-CoV-2 pandemic impacted access to inpatient rehabilitation services. At the current state of research, it is unclear to what extent the adaptation of rehabilitation services to infection-protective standards affected patient-centered care in Germany.The aim of this study was to determine the most relevant aspects of patient-centered care for patients in inpatient rehabilitation clinics under early phase pandemic conditions.A deductive-inductive framework analysis of online patient reports posted on a leading German hospital rating website, Klinikbewertungen (Clinic Reviews), was performed. This website is a third-party, patient-centered commercial platform that operates independently of governmental entities. Following a theoretical sampling approach, online reports of rehabilitation stays in two federal states of Germany (Brandenburg and Saarland) uploaded between March 2020 and September 2021 were included. Independent of medical specialty groups, all reports were included. Keywords addressing framework domains were analyzed descriptively.In total, 649 online reports reflecting inpatient rehabilitation services of 31 clinics  were analyzed. Keywords addressing the care environment were most frequently reported (59.9%), followed by staff prerequisites (33.0%), patient-centered processes (4.5%), and expected outcomes (2.6%). Qualitative in-depth analysis revealed SARS-CoV-2–related reports to be associated with domains of patient-centered processes and staff prerequisites. Discontinuous communication of infection protection standards was perceived to threaten patient autonomy. This was amplified by a tangible gratification crisis of medical staff. Established and emotional supportive relationships to clinicians and peer groups offered the potential to mitigate the adverse effects of infection protection standards.Patients predominantly reported feedback associated with the care environment. SARS-CoV-2–related reports were strongly affected by increased staff workloads as well as patient-centered processes addressing discontinuous communication and organizationally demanding implementation of infection protection standards, which were perceived to threaten patient autonomy. Peer relationships formed during inpatient rehabilitation had the potential to mitigate these mechanisms. In modern health systems, the relevance of patient-centered care (PCC) continues to progress as it is associated with improved patient satisfaction, self-management, and perceived quality of care ,2. The SIn Germany, approximately 85% of medical rehabilitation services are provided in inpatient care settings . The cenDue to a historically grown separation of acute care and medical rehabilitation, the German system faces declining trends of rehabilitation claims as patients are self-responsible to initiate application processes to IRS and intersection communication among health care sectors is fragmented . Since pLooking at other inpatient care settings, Andersson et al  investigConsidering limited or delayed admission to IRS, changed process parameters, reduced availability of services, and nontransparent longitudinal leadership structures, it is unclear to what extent these adaptations affected PCC during rehabilitation in Germany. Moreover, an appropriate inclusion of patient perspectives is currently pending. Thus, the particular interest of this study was to evaluate which aspects of PCC were important for IRS recipients during the early phase of the SARS-CoV-2 pandemic in Germany. In that regard, the following research question motivated this study:Which aspects of PCC are relevant for patients in inpatient rehabilitation clinics and how do they evaluate these aspects to be achieved under conditions of the SARS-CoV-2 pandemic?By identifying SARS-CoV-2–related aspects affecting PCC in inpatient rehabilitation, the research team aimed at informing rehabilitation clinics to not only become resilient health care organizations but also to meet patient needs in highly demanding and exceptional circumstances of the future. Despite stating palpable organizational interests, this ambition also reflects a moral attitude being of central relevance to any health care organization.In this qualitative analysis, a deductive-inductive framework approach was used. The applied framework was developed by Liu et al  aiming aThe sample of online posted patient reports was guided by a theoretical selection process. Reports on hospital stays posted on the most commonly used German hospital rating website Klinikbewertungen (Clinic Reviews)  were inc2  Even after talking to doctors, nothing has really changed. All in all, I have mixed feelings and it is very important not to blame everything on Corona.Oncological rehabilitation clinic, BrandenburgDespite availability issues, it became apparent that hygiene legislations were more likely to be accepted if they were easily integrated into organizational routines. This was also seen as having the advantage to create a more familiar environment as, for instance, therapeutic care groups decreased in size. One factor not directly attributable to the pandemic was the available food, which was perceived to be inconsistent with nutrition education events offered during rehabilitation.Due to the Corona pandemic, procedures were changed which wasn’t only bad: For instance, I perceived the cutting of the reference group actually very pleasant and more personal. I perceived the sessions to be more intense and individual. Perhaps, it should be considered whether this can be maintained after the pandemic.Psychiatric rehabilitation clinic, BrandenburgIn general, the domain of expected outcomes was of minor significance for patients in German rehabilitation clinics. However, it became apparent that a distinct communication of patient-relevant outcomes and their respective change after rehabilitation was positively associated with patient satisfaction. The communication of changes in outcomes seemed to be more straightforward to be implemented in somatic care facilities. Furthermore, available emotional support during IRS was perceived to facilitate the individual healing process by having a direct impact on activating self-efficacy and self-management potential.I want to compliment the care provided by doctors and therapists. I arrived here with severe swelling and effusion in the knee and leave the rehab with great mobility and stability in my joint .Orthopedic rehabilitation clinic, SaarlandI arrived as a diabetic with overweight, having taken medication for three years, including for high blood pressure. The holistic care of the staff has resulted in, me losing 12 kilograms in four weeks and I am now medication free. My long-term blood sugar now is 5.9 and I’m coming from over 8.Orthopedic-diabetic rehabilitation clinic, BrandenburgThe domain of “peer relationship” inductively emerged during the process of analysis. Empowering peer-to-peer relationships was valued to have the potential to mitigate adverse effects of the pandemic on IRS. Thus, some patients reported that their stay remains unforgotten mainly due to their peers, who compensated for negative inconveniences. Patients also appealed to the personal responsibility of their peers. In their understanding, only active engagement allows expectations of rehabilitation success.A rehab is not a vacation, your own participation is expected and necessary- success depends on you and your attitude toward rehab and your own illness; a rehab facility is not a hotel with many stars…Psychiatric rehabilitation clinic, BrandenburgThe first week was shaped by uncertainty of the unknown, but the great people I met supported me to deal with the problems that arose. Usually, we would meet for lunch to tell each other what had happened during the day. Many times, we were just listeners when a colleague of ours was feeling bad.Psychiatric rehabilitation clinic, BrandenburgA comprehensive summary of online reported patient experiences addressing PCC domains and attributes is provided in For patients receiving IRS, aspects of the care environment, staff prerequisites, and patient-centered processes were predominantly relevant to evaluate their inpatient stay. SARS-CoV-2–related adaptation mechanisms affecting these domains comprised discontinuous communication and elaborate implementation of infection protection standards, which were perceived to threaten the personal autonomy of action. These mechanisms were amplified by tangible gratification crises of medical staff. However, the prevalence of established and emotional supportive relationships to clinicians and peer groups provided the potential to mitigate the adverse effects of hygiene protection standards on IRS. Moreover, a distinct communication of therapeutic outcome variation seemed to support patient satisfaction. These insights provide the opportunity to develop informed strategies fostering resilient organizations that sustainably embody PCC within the setting of rehabilitative care.Our findings are partly in line with those of Liu et al  who demoConsidering the suitability of the applied PCC framework , this anIn this analysis, patients perceived a significant number of medical staff to present aspects of a developing gratification crisis reflecting generic psychological distress. This finding is supported by Dobson et al , who ideDespite illustrated challenges of IRS during the early phase of the SARS-CoV-2 pandemic, most patients felt safe and supported infection protection standards. This is underpinned by a survey of oncological patients treated in German rehabilitation clinics, 87% of whom reported to feel safe in facilities . AlthougFirst, one limitation of our study is the limited representativeness of findings for other rehabilitation settings within Germany as hygiene regulations differed across states. Moreover, online reported patient complaints as a scientific data source produce concerns of representativeness and subjectivity as sample characteristics are uncontrolled and widely unknown. However, an analysis of Facebook reviews demonstrated that contents of reviews do not correlate with inpatient quality assessment indicators but instead correlate with a standardized national survey of patient experiences in German obstetrics . MoreoveSecond, using online reported patient complaints is accompanied by unknown sample characteristics and thereby associated with hazards of selection bias. In this context, Han and colleagues  identifiMoreover, research activities of economic sciences identified online product ratings to be influenced by social dynamics. It is acknowledged that product ratings are not only affected by individual experiences but rather by prior ratings of one’s peer group ,38. At tAlong with these stated limitations, this analysis faces unique restrictions. As the distribution of reports across included clinics varied strongly, a cluster bias of included rehabilitation clinics with disproportionately strong patient rating activities of some facilities cannot fully be ruled out. In this regard, it will be of interest to further investigate which clinic-related parameters affected ratings of clinics with above-average report numbers. Additionally, the current selection of keywords reflecting PCC domains was made inductively and potentially implies an incomplete list of keywords supporting a distortion of distributed domains.Despite these limitations, patient rating portals became increasingly popular over the last 10 years ,39, whicTaking the present results into account, future research direction should investigate country-specific differences in the perceived significance of PCC domains. For instance, it remains to be answered why online reports of German inpatient care recipients are currently dominated by reports about the care environment, whereas health-relevant outcome expectations seem to have a subordinate role.This analysis reflects previous research as German patients predominantly reported feedback associated with the care environment. SARS-CoV-2–related reports were strongly affected by aspects of patient-centered processes addressing discontinuous communication and an organizationally demanding implementation of infection protection standards, which was in some cases perceived to threaten patient autonomy. This perceived threat in reduced autonomy was amplified by a tangible increase in staff workload. Developed peer relationships during the rehabilitation stay had the potential to mitigate these mechanisms.


Over the past few decades, advances in electroencephalography (EEG) recordings and brain stimulation has permitted an unprecedented view of how specific brain structures communicate as well as organize complex cognitive functions. Specifically, neurotransmitters  have all been shown to have an impact on neural oscillations throughout the brain, linking them to changes in cognitive functions such as memory, attention, and executive function. While these interactions are still widely unexplored, their appearance in neurological disorders through cross-frequency coupling (CFC) brings light to the vital role they play in orchestrating healthy brain function. This brief review serves to highlight the important role each neuromodulatory system plays in changing widespread neural networks, emphasizing their involvement in health and disease to help inform more translational brain stimulation technologies.Using EEG and local field potentials (LFPs) as an index of large-scale neural activities, research has been able to associate neural oscillations in different frequency bands with markers of cognitive functions, goal-directed behavior, and various neurological disorders. While this gives us a glimpse into how neurons communicate throughout the brain, the causality of these synchronized network activities remains poorly understood. Moreover, the effect of the major neuromodulatory systems  on brain oscillations has drawn much attention. More recent studies have suggested that cross-frequency coupling (CFC) is heavily responsible for mediating network-wide communication across subcortical and cortical brain structures, implicating the importance of neurotransmitters in shaping coordinated actions. By bringing to light the role each neuromodulatory system plays in regulating brain-wide neural oscillations, we hope to paint a clearer picture of the pivotal role neural oscillations play in a variety of cognitive functions and neurological disorders, and how neuromodulation techniques can be optimized as a means of controlling neural network dynamics. The aim of this review is to showcase the important role that neuromodulatory systems play in large-scale neural network dynamics, informing future studies to pay close attention to their involvement in specific features of neural oscillations and associated behaviors. Neural oscillations are thought to be an essential driver of interaction, communication, and information transmission throughout the brain ,2,3. EvoThe neuromodulatory systems, including the noradrenergic, cholinergic, and dopaminergic systems, play a pivotal role in the regulation and synchronization of neural oscillations. These systems provide direct axonal projections to most structures of the brain, regulating various brain functions through the release of neurotransmitters ,23,24,25This review will focus on how these three neuromodulatory systems can individually modulate neural oscillations, looking specifically at how their activation can change large-scale neural network synchrony in cognitive functions and neurological disorders. This phenomenon has yet to be fully understood, and looking at how each system’s activation or inhibition affects large scale oscillatory patterns may help uncover the origin and causality of complex behaviors and neurological disorders. These insights will hopefully inform a new direction of research that looks to further investigate how neuromodulation could improve and/or shape brain functions through changing large-scale neural oscillations.Over the past few decades, an abundance of research has been conducted exploring the role different frequency bands play in representing the neural signals behind cognition, behavior, and neurological disease. These have been characterized by abstracting the frequency spectrum into five unique frequency bands: Delta (1–3 Hz), Theta (4–7 Hz), Alpha (8–12 Hz), Beta (13–30 Hz), and Gamma (30–100 Hz) . While aThroughout normal cognition, each oscillatory frequency band has been linked to a specific behavioral correlate or executive function. Generally speaking, delta waves are associated with a wide variety of cognitive functions. Studies have shown that the delta oscillatory phase is correlated with the reaction time of behavior, playing a role in the synchronization of neural populations across multiple brain regions . AdditioWhile each frequency band is instrumental to maintain healthy brain function, neurological diseases seem to alter specific oscillatory properties, oftentimes leading to cognitive dysfunction. To begin, abnormal delta oscillations have often been linked to neurological conditions associated with sleep. Specifically, non-rapid eye movement sleep in both Schizophrenia and Alzheimer’s disease has been associated with a depressed delta power when compared to healthy patients ,55 CliniWhile each frequency band seems to dissociate into a wide variety of behaviors or cognitive functions, the synchrony between each band through cross-frequency coupling (CFC) analysis has brought new light to the complexity of neural networks . Recent Previous studies show more instances of CFC playing a role in cognitive functions across memory, learning, attention, and decision making. Specifically, alpha–beta phase–amplitude coupling (PAC) has been seen synchronized in the medial prefrontal cortex during decision making, as well as the phase of delta and theta bands, suggesting the role of PAC in feedback coding ,79. In dCFC is also thought to be the brain’s method of transferring large amounts of information across distant regions of the brain in an organized fashion, and it has been shown to impact different forms of learning and memory across species. Theta oscillatory synchronization between the prefrontal cortex (PFC) and the amygdala supports communication across interneuron networks and is associated with fear learning . Theta bIn playing such a large role in neural physiology, it is no surprise that CFC has also been involved in a variety of neurological diseases. Being most observed in working memory studies, PAC has been associated with diseases including schizophrenia, obsessive–compulsive disorder, Alzheimer’s disease, epilepsy, and Parkinson’s disease. For example, it has been suggested that CFC may play a role in Parkinson’s disease as excessive PAC between beta and gamma bands has been observed in advanced-stage patients with movement deficits ,88. AbnoCross-frequency coupling has been found in the synchronization across subcortical and cortical regions, orchestrating a bottom-up circuit where deep brain regions such as the hippocampus and neuromodulatory centers such as the locus coeruleus (LC), ventral tegmental area (VTA), and basal forebrain (BF) have direct control of brain-wide neuronal synchrony. However, this phenomenon has yet to be explored in patients, as invasive stimulation and optogenetic/chemogenetic manipulations are challenging to conduct in human studies. Future experiments using animal disease models would be better positioned to look into how specific brain structures modulate CFC to help uncover the causal relationship between cognitive abilities and neural oscillations in health and disease.The noradrenergic system plays a very important role in the modulation and regulation of norepinephrine (NE) throughout the brain. The center of norepinephrine creation, the locus coeruleus, is a complex nucleus with projections to almost every area of the cortex and subcortical areas ,95,96,97The cholinergic system, like the noradrenergic system, also plays a vital role in the control of neural oscillations throughout the brain and cortical structures . StudiesLike noradrenergic modulation, cholinergic modulation has not specifically been studied in conjunction with cross-frequency coupled phenomena. Instead, studies utilizing widespread neural recordings have looked to uncover the direct role of ACh in behavior and erratic firing patterns associated with neurological disorders. Specific low-frequency oscillations including delta, theta, and alpha have been seen to be suppressed by brainstem cholinergic neurons, while high-frequency bands such as beta and gamma are accompanied by an increased release of ACh in the thalamus and cortex . SupportOftentimes, Ach-triggered neural synchrony has been explored using activation or inhibition studies through optogenetics and pharmacological agents. Specifically, phasic optogenetic stimulation of the basal forebrain has been seen to modulate the cortical topography of auditory steady state responses, with phase-locked stimulation enhancing the power of cortical responses and increasing their synchronization, as well as changing broadband gamma power ,138,139.The dopaminergic system is critically important to the circuitry and control of cognitive functions in the prefrontal cortex . Like thThe dopaminergic system has been shown to be heavily involved in the shaping of neural oscillations in a variety of cognitive functions and neurological disorders. Studies utilizing electrophysiology recordings have demonstrated the vital role of dopamine activation throughout the brain. Specifically, dopaminergic receptor activation has been linked to weaker alpha and beta oscillations in the PFC, while depletion of dopamine has been linked to increased power in beta oscillations in the cortex and subthalamic nucleus of rats ,146. DopThe dopaminergic system clearly acts as a controller of large-scale neural oscillations, but its causal relationship with CFC is largely unknown. Looking at dopamine-triggered synchronization studies may help provide context to the role of dopamine in CFC. In Parkinson’s patients, chronic dopaminergic transmission interruption promotes excessive cortical beta synchronization that is seen in high coherence between motor and somatosensory cortical activities . DesynchWhile future research needs to be conducted exploring each neuromodulatory system’s direct role in neural oscillations, looking at another non-invasive metric, such as the pupil, could provide insight into the interaction between neuromodulation and cognition. Previous work has showcased the ability for the pupil to be used as a non-invasive readout of the central arousal state , with possible involvement of the noradrenergic and cholinergic systems ,105,156.The functional outcomes of understanding the unique role each neuromodulatory system plays in mediating cross-frequency coupling to facilitate certain brain functions have immense potential. Already, researchers have been able to develop neural interfaces to control and modulate a variety of neurological pathways . More moWhile these methods are promising, VNS is proving to be one of the most effective approaches to non-invasive neuromodulation. The vagus nerve projects to a variety of brain regions, including neuromodulatory centers such as the LC, and VNS has been shown to be involved in phase–amplitude coupling ,182. TheFuture work reliably assessing real time cross-frequency coupling relationships in EEG and LFP signals could provide insight into how stimulation techniques can properly modulate cognitive function and behavioral outcomes. Experiments looking at key EEG features such as phase, amplitude, power, and frequency should be conducted in order to further explain how neural oscillations change in response to different cognitive processes. Selective neuromodulation of the noradrenergic, cholinergic, and dopaminergic system may provide insight into how different CFC phenomena occur, guiding our understanding into how complex synchronous neural networks communicate and formulate complex circuits to direct cognitive functions . Closed-The role of the neuromodulatory systems in influencing neural oscillations is an understudied topic. Decades of research have helped bring to light the relationship between neural oscillations and higher cognitive functions, but the causality behind these signals remains poorly understood. By reviewing the literature of each of the three major neuromodulatory systems  and their representative cortical projections, effects on neural oscillations, and involvement in cross-frequency coupling, the complex nature of these neural computations can hopefully be further explained. To enhance our understanding of this relationship, it is essential to look towards specific oscillatory features such as cross-frequency coupling to understand the intricacies within large-scale neural synchronization. Studies emphasizing the importance of the activation and inhibition of these neuromodulatory centers in conjunction with frequency domain analysis could prove critical to understanding the causality of different behaviors and neurological disorders. These results could help inform the next generation of closed-loop neural stimulation devices with effective real-time outcomes, prioritizing the role of neuromodulation in controlling large-scale neural oscillations to restore or enhance brain functions.


RNA has emerged as a revolutionary and important tool in the battle against emerging infectious diseases, with roles extending beyond its applications in vaccines, in which it is used in the response to the COVID-19 pandemic. Since their development in the 1990s, RNA interference (RNAi) therapeutics have demonstrated potential in reducing the expression of disease-associated genes. Nucleic acid‐based therapeutics, including RNAi therapies, that degrade viral genomes and rapidly adapt to viral mutations, have emerged as alternative treatments. RNAi is a robust technique frequently employed to selectively suppress gene expression in a sequence-specific manner. The swift adaptability of nucleic acid‐based therapeutics such as RNAi therapies endows them with a significant advantage over other antiviral medications. For example, small interfering RNAs (siRNAs) are produced on the basis of sequence complementarity to target and degrade viral RNA, a novel approach to combat viral infections. The precision of siRNAs in targeting and degrading viral RNA has led to the development of siRNA-based treatments for diverse diseases. However, despite the promising therapeutic benefits of siRNAs, several problems, including impaired long-term protein expression, siRNA instability, off-target effects, immunological responses, and drug resistance, have been considerable obstacles to the use of siRNA-based antiviral therapies. This review provides an encompassing summary of the siRNA-based therapeutic approaches against viruses while also addressing the obstacles that need to be overcome for their effective application. Furthermore, we present potential solutions to mitigate major challenges. Researchers have investigated siRNAs targeting vital proteins involved in the transcription/translation processes, including viral VP24, VP35, and polymerase L –48. DunnDesigning RNAi strategies for combating HIV involved greater complexity due to the well-documented challenges. The difficulty in treating HIV-1 infection is attributed to the rapid virus replication rate and its propensity to swiftly escape or the emergence of resistant mutants in response to antiretroviral therapy .FurthermThe US Food and Drug Administration approved a phase I clinical trial in May 2007 to evaluate RNAi as a nucleic acid therapy using a lentiviral vector capable of infecting nondividing cells for HIV-1 infection . HoweverThe practical application of siRNA for therapeutic purposes holds significant potential and is supported by its proven effectiveness for gene silencing precision, as demonstrated in specific in vitro and in vivo studies. Nevertheless, why have we not yet witnessed the approval of antiviral siRNA two decades after its discovery? The answer lies in the numerous barriers Fig. , discussTheoretically, as previously mentioned, siRNA modalities can be designed and targeted to silence any viral gene of interest, which makes them efficient tools for a broad range of antiviral applications . DespiteThere are various factors that impact the effectiveness of siRNA, such as the guanine‐cytosine (GC) content, nucleotides at siRNA termini, thermodynamic properties, siRNA structure, and accessibility of a target site , 55. To One of the aforementioned primary challenges, the maintenance of siRNA stability is a significant obstacle to desired therapeutic efficacy of siRNA treatments.The foremost extracellular hurdles associated with antiviral siRNA therapy include rapid systemic clearance, rendering siRNAs susceptible to nucleases-mediated degradation. Unmodified RNA molecules are prone to degradation by extracellular nucleases, and their negatively charged nature hampers their ability to traverse the hydrophobic cytoplasmic membrane, negatively affecting both their stability and pharmacokinetics –64. ThesDue to their negative charge, naked or otherwise unmodified siRNAs cannot penetrate the cell membrane, which consists of a negatively charged bilayer of phospholipids and functional proteins . MoreoveOnce internalized, siRNAs must then escape from endosomal compartments and reach the RNAi machinery. Endosomal trapping is another significant obstacle to the success of RNAi-based therapy and has been extensively covered in several excellent reviews –71. CellA way to enhance siRNA stability is to use different types of 2′ sugar modifications; for example, fluorine substitutions, which can increase resistance to endonucleases, can be incorporated into the delivery system . ModificThe delivery of antiviral siRNA is a significant challenge to siRNA therapeutics development for human use, largely due to the pharmacological properties of siRNA , 80, 81.Using siRNA delivery vehicles to protect against degradation in the circulatory system is critical for practical siRNA-mediated silencing . NanocarConsideration should also be given to direct injection of antiviral siRNA into infected tissues for cell-specific targeting . To mitiDelivery systems for antiviral siRNAs can be classified into vectors and nanoparticles , 88. PlaBoth lipid- and polymer-based systems share key chemical properties for successful delivery. Polymer-based siRNA delivery vehicles offer structural flexibility and nuclease protection . CationiInorganic nanoparticles consisting of calcium phosphate, gold, carbon, and iron oxides transport siRNAs effectively due to their small size and high permeability compared to the those of liposomes and polyplexes. Effective delivery systems can be designed on the basis of the elements, bonds, and functional groups necessary. The effectiveness, stability, and precise sequence complementarity of siRNA can be enhanced by synthesis with modified nucleotides. The components of short-stranded siRNA systems also include hydrophobic modification marks and tertiary amines, and these systems show the ability to interact with short siRNA strands through various binding interactions.Future improvements to antiviral siRNA delivery efficacy may involve (1) the use of new targeting ligands and chemical probes that specifically bind to surface markers on infected cell populations, (2) increasing the efficacy of siRNA uptake into the cytoplasm, (3) developing materials with low toxicity to widen the antiviral therapeutic window, (4) designing materials with defined degradation products that can be metabolized for repeated dosing, (5) simplifying the formulation procedure, and (6) exploring delivery to organs other than the liver –101.Many issues must be considered when siRNAs are designed, including sequence space conservation, the siRNA sequence, and siRNA and target mRNA structure constraints . CombiniDespite extensive in silico efforts, unintended siRNA activity is often observed in vitro and in vivo, and extensive preclinical testing of siRNAs in a variety of in vitro, ex vivo, and in vivo models with complex genomes is critical . UnfortuFor example, unintended suppression of nontarget genes, also known as "off-target effects," can cause difficulties in data interpretation and may induce toxicity. In addition to the specificity and efficiency of the silencing potency of siRNAs, pharmacodynamic-related problems include off-target RNAi activity and immunotoxicity triggered by antiviral siRNA carriers . It is iThe side effects of RNAi-based drugs can result from multiple factors, including off-target effects –110. TheDuring the design of siRNAs, homology and specificity analyses should be performed to prevent off-target effects  because Because the antisense strand in siRNA guides the RISC, precise sequence complementarity of this strand is pivotal for on-target RNAi and minimized off-target effects . This unOff-target analysis should lead to the exclusion of siRNAs with potential binding affinity for human mRNAs. Recent work by Fakhr et al. provides comprehensive insights into sequence selection using specific protocols  or compuO-methyl ribose substitution at position 2 in the guide strand, was developed to reverse the silencing of partially complementary transcripts [O-methyl modifications to the seed region, which consists of nucleotides 2–8 on the antisense strand, has been shown to provide protection against endonuclease activity and prevent off-target effects [O-methyl chemical and locked nucleic acid modifications of nucleotides in the seed region (positions 2–8) of the siRNA guide strand significantly reduced seed-matched off-target effects ['-O-methyl modifications into the siRNA seed region. These two types of chemical modifications act through distinct molecular mechanisms to suppress off-target effects [A particular position-specific chemical modification of siRNA, 2′-nscripts , 123 or nscripts , 125. Fo effects . Song et effects , showing effects . Yoshiak effects . Their r effects .In recent findings, Shiohama et al. proposed that modifying the sense strand with X or Z moieties eradicated off-target effects caused by complementarity of the sense strand without impeding siRNA silencing efficiency . Varley Ideally, siRNAs would show absolutely specificity, thereby regulating only the target gene of interest. However, an increasing body of evidence suggests that this is not necessarily the case , 132. ImSynthetic siRNA duplexes have been shown to have immunological effects that can induce high levels of inflammatory cytokine and type I interferon production . These rThe activation of the innate immune system by siRNAs is mediated through diverse mechanisms involving recognition by both cytoplasmic and endosomal receptors, including TLRs , 137. ToThe difficulties associated with siRNAs triggering immune responses, leading to inflammation and toxicity, remain a significant obstacles to their application as gene expression regulators. Recent findings indicate that siRNAs can also influence the translation of ectopically transcribed genes through partial complementarity, causing nonspecific interferon responses . siRNAs Unmodified or improperly formulated siRNA may activate TLR3 and adversely affect the blood and lymphatic systems . NonmodiO-ribose methylation modification have been developed [To mitigate the effects of immunostimulatory siRNA sequences, ribose modifications are commonly employed, as they effectively reduce cytokine production . For exaeveloped .O-methyl modifications into both sense and antisense strands diminishes RNase recognition [The incorporation of 2′-ognition , 148. Thognition , effectiognition . Substitognition , 151.Delivering synthetic siRNA at high doses has been shown to induce cytotoxic interferon and inflammatory cytokine secretion in a sequence-binding-dependent manner . MoreoveSince the RNAi pathway is a catalytic pathway and a single siRNA molecule can therefore bind to and regulate multiple mRNA copies, the administered dose can be reduced to reduce off‐target activity-associated toxicity . FurtherAn important consideration in developing antiviral RNAi therapies is the tendency of viral genomic sequences to undergo mutations that enable them to evade host immune responses. This creates problems in developing effective antivirals and vaccines, especially against RNA viruses such as HIV-1 and hepatitis C virus, which exhibit high mutation rates. In the early stages of RNAi therapy development, a single siRNA sequence was commonly used to achieve effective gene silencing. However, recent advancements have addressed viral mutations by employing multiple siRNAs. Combining siRNAs to simultaneously target several viruses and/or host targets mitigates the potential loss of effectiveness resulting from mutations at a specific target site.There are two major obstacles in addition those mentioned above that must be overcome for siRNAs to become effective antiviral agents: (i) the short duration of antiviral activity due to the emergence of escape mutants resistant to siRNA, as long-term silencing of viral protein expression by siRNAs has been reported to result in the emergence of viruses resistant to RNAi , 155 andThe selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. RNAi-resistant viral mutants emerge rapidly, mainly in targeted viral sequences. Previous studies describing resistant escape mutants to siRNA treatment suggest that single point mutations can diminish or even abolish the RNAi effect –157.As mentioned above, target sequences that are highly conserved among virus isolates should be selected. One of the practical strategies to address both the problems of sustained silencing and the emergence of escape mutants is to design siRNA based on viral RNA sequences that are conserved and invariant across various strains , 159. HoFurther systematic investigations are required to identify the appropriate conserved target sites for siRNAs, thereby ensuring their universal and lasting antiviral effects. Notably, von Eije et al. observed that viral escape was profoundly restricted by the selection of highly conserved targets for their therapeutic strategy .Westerhout EM et al. demonstrated that viral mutations induce alternative folding of the RNA structure that can occlude a target sequence and prevent siRNA binding, resulting in a reduction in RNAi efficiency . BecauseIt has been suggested that RNAi efficiency is affected by the accessibility of the target RNA, which may be influenced by protein binding and the formation of RNA secondary structure –167. RNAKonishi et al. hypothesized that viral resistance to antiviral siRNAs may be due to low siRNA delivery efficiency or inaccessibility of the target genome by the siRNA molecule, suggesting that multitarget siRNA molecules may be more effective by lowering the likelihood that simultaneous mutations at multiple sites affect RNAi efficacy .Jackson and colleagues suggested that using multiple siRNA duplexes to silence the target gene increased the likelihood of observing the desired phenotype and expression pattern . This apMost insect or plant RNA viruses have evolved RNA silencing suppressors to counteract antiviral RNAi effects. Among plant viruses, the tombusvirus-encoded P19 protein blocks RNAi machinery by binding siRNAs via their dsRNA-binding domain, thereby sequestering siRNAs and preventing their action in RNAi pathways and preventing siRNA incorporation into the RISC , 175. SiAlthough the majority of RNA silencing suppressors have been identified in plant and invertebrate viruses, an increasing number of mammalian viruses have also been found to encode suppressors. RNA silencing suppressor proteins include the nucleocapsid protein of SARS-CoV-1  and SARSCui et al. provided evidence showing that the nucleocapsid protein of SARS-CoV efficiently inhibited Dicer-mediated dsRNA cleavage and post-Dicer activity by sequestering dsRNAs and siRNAs . The autFurthermore, Haasnoot et al. showed that adenovirus virus-associated RNAs inhibit RNAi by acting as decoy substrates for Exportin 5, Dicer, and the RISC , 196. EbAlthough viral RNA silencing suppressors may counter RNAi effects in the context of natural infections, these factors may not necessarily diminish the effectiveness of siRNA-based antiviral therapeutics . InteresThe use of siRNA to silence genes has emerged as a powerful approach for studying cellular processes and targeting disease-causing factors with precision. Over the past twenty years, there have been notable breakthroughs in the development of siRNA-based therapeutics for a range of diseases, including viral infection. RNAi is a gene silencing mechanism that provides a powerful means to specifically inhibit viral infection. Increasing evidence has emerged to suggest that RNAi pathways are evolutionarily conserved defense mechanisms against pathogenic viral infections , providiTo enhance the cellular uptake of siRNAs, synthetic nanoparticles made up of polymers, lipids, and conjugates can be employed, along with the integration of cell-specific targeting ligands in carriers . For exaTo use siRNA as an antiviral therapy, side effect concerns, including off-target effects, must be addressed. Although it is possible to design virus-specific siRNAs that do not cross-react with the human genome, this possibility needs to be confirmed. There is a risk of side effects if an siRNA partially hybridizes with untargeted mRNAs, so a balance between potency and safety must be considered when formulating the final product. In vivo studies are necessary to investigate any potential inflammatory responses to antiviral siRNA treatment. Given the constantly evolving viral genome, it is important to target specific and highly effective therapeutics at conserved genomic regions to inhibit viral replication . In addiSelectively silencing genes by hijacking the endogenous RNAi pathway with exogenous antiviral siRNAs has become a widely used strategy to study gene function, and further, this approach has shown impressive therapeutic potential that is expected to be realized soon. The biotechnology industry has devoted significant resources to the development of siRNA therapeutics for treating various diseases, including viral infections, and an increasing number of studies have focused on the development of antiviral siRNAs in recent years. Despite obstacles to the use of siRNA antiviral therapy, it is anticipated that the future of RNAi therapeutics is bright because of interdisciplinary efforts and technological advancements. Therefore, it can be projected that antiviral siRNA applications will provide enormous potential for the treatment of viral infections in the future.


Early buzz around ChatGPT’s  proficieEarly conversations about LLMs in healthcare tended towards extremes, either lauding their breathtaking potential or lamenting the threat they pose to human-centric care. More recently, nuanced narratives regarding the ethical implications of LLMs have started to emerge ,8. Stilldevelopment of LLMs are widely discussed, with substantial dialogue dedicated to intrinsic equity and general ethical considerations of how, why, and with what data and algorithmic design choices these models should be developed [deployment are less commonly discussed. These emergent risks arise from pragmatic use by end users  in real-world settings. It is critical to understand emergent equity risks of LLMs to avoid replicating inequities that resulted from the use of other technologies in medicine [Equity risks originating in the eveloped ,11. Equimedicine .For patients, LLMs have a remarkable ability to generate human-like responses and summarize complex or hard-to-find information ,4, proviFor clinicians, LLM-supported tools offer efficiency gains that may help address issues of burnout and dissatisfaction. For example, DocsGPT  automateFor healthcare systems, LLM-supported technologies offer large-scale automation of patient communication, increasing customer relationship management efficiency and potentially improving value-based care metrics; these tools could also enable novel strategies to address health disparities, such as proactively connecting patients with identified social vulnerabilities with relevant resources. Unfortunately, competing priorities in care delivery for healthcare systems (eg revenue generation) too often result in the deprioritization of health equity efforts, including in the use of novel technologies. For example, as healthcare systems experiment with charging patients for messaging providers, there is a risk of minoritized patients preferentially turning to search engines and chatbots for medical advice. Healthcare systems that adopt widespread direct-to-patient LLM strategies that remove clinicians entirely from patient exchanges also risk impersonal, inaccurate, or biased –16 commuFinally, LLM products that organizations purchase for clinicians will contain behind-the-scenes prompting that shape how equitable responses are. While end users of AI models have traditionally had little power to alter the outputs of the models they’ve used, prompting has changed that dynamic . PromptiUltimately, emergent equity risks in LLM deployment are complex and may be difficult to anticipate as novel use cases arise. Organizations such as the White House and National Academy of Medicine are working toward comprehensive frameworks for the ethical use of AI. However, in the meantime healthcare systems have a responsibility to engage in iterative risk mitigation around health equity as they rapidly deploy LLMs. Recommendations for addressing emergent equity issues in LLM deployment include:Evaluating vendors can be facilitated by equity-informed requests for proposals or other intake tools designed to probe for equity-related concerns in product development, key performance indicators, and internal values. For LLM-supported products, this would include questions regarding the source and diversity of the training data (e.g. ), procesWhen using LLMs in practice, healthcare systems should establish robust post-deployment monitoring protocols for disparities in access, utilization, or outcomes. Additionally, they should work with vendors to regularly monitor model responses, both by humans trained to systematically evaluate for bias and by models trained to detect inequitable responses (e.g. responses that more often tell women that their reported symptoms are likely psychogenic than men). Patients and clinicians should retain the option to report biased responses at any time. Epic has created a forum for healthcare systems to share the GPT prompts that they are using with each other . This foWhile some healthcare systems may rely entirely on out-of-the-box prompt libraries (e.g. DocsGPT ), most wLLMs are being rapidly operationalized, with tremendous potential to improve healthcare access, satisfaction, and outcomes. However, they also have the potential to amplify inequities by promoting digital health segregation and reinforcing underlying biases in the digital world. We must break the cycle of addressing health equity issues only after harm has been done and deliberately center equity in large language model deployment before it is too late.


In his Nobel lecture upon receiving the Nobel Prize in Physiology or Medicine in 1945, Sir Alexander Fleming made a remarkably prescient prediction regarding the potential repercussions of the improper use of penicillin. He cautioned that such misuse would inevitably result in antibiotic resistance and treatment failures. Now, after several decades have elapsed, we find ourselves grappling with a shortage of effective options in antibiotic arsenals when confronted with numerous perilous pathogens. Projections indicate that if the current trend of antibiotic resistance prevalence continues to escalate, and if no new efficacious therapies are devised, an estimated 10 million deaths worldwide could occur due to superbug infections by 2050.Effectively addressing this challenge necessitates a comprehensive understanding of the underlying mechanisms of resistance, as well as the development of innovative strategies to combat antimicrobial resistance (AMR). Recent advancements in single-cell tools have yielded valuable insights into the heterogeneity of microbial populations and offered a significant advantage in reducing the turnaround time (TAT) in current clinical diagnostic protocols. This is achieved by circumventing the need for protracted cultivation processes, which can take several days or even weeks to yield definitive results. Single-cell methodologies expedite the diagnostic process substantially, allowing for prompt intervention and targeted treatment.Xu et al. demonstrated the rapid  and accurate (100%) classification of 94 clinical isolates resulting from fungal infections, reporting a sample-to-result time within 1 h. Lu et al. reported signature changes in the Raman spectra of 12 species of pathogenic bacteria, as well as in antibiotic-sensitive and resistant strains of Acinetobacter baumannii, with a higher nucleic acid/protein ratio observed in the sensitive strains.Among these methodologies, single-cell spectroscopic techniques, specifically Raman spectroscopy and optical photothermal infrared (O-PTIR) spectroscopy, have garnered particular attention in this Research Topic. Raman and IR spectroscopic techniques can generate highly specific vibrational fingerprints of biomolecules, providing a snapshot of the presence and phenotypic identity of pathogens. By joining vibrational spectroscopy with advanced computational methods, researchers can unravel the intricate relationship between molecular signatures, thereby establishing the foundation for the rapid identification of pathogens at the single-cell level. Wang et al. further refined the current Raman-DIP approach by incorporating a high concentration of sodium acetate as an inhibitor of pathogen replication, while simultaneously preserving their metabolic activity. This breakthrough allowed for the rapid quantification of viable bacteria in urinary tract infections, within a timeframe of <3 h.Beyond pathogen identification, an elegant approach to measuring microbial metabolism under the influence of antibiotics via Raman spectroscopy involves culturing cells in partially deuterated water. As metabolically active cells incorporate deuterated water molecules into new biosynthetic products, they create new carbon–deuterium (C–D) bonds. This technique, known as Raman-deuterium isotope profiling (Raman-DIP), has proven to be remarkably swift, with fast-growing cells requiring <20 min in deuterated water to exhibit active metabolism. In their study, Shams et al. were the first to incorporate DIP into O-PTIR spectroscopy, thereby showcasing the characteristic C-D stretching at 2,159 cm−1 and detecting AMR at the single-cell level.In contrast to single-cell Raman modalities, research utilizing IR spectroscopy has predominantly been conducted at a population level, primarily due to limitations imposed by the diffraction limit when employing longer wavelengths. Recent advances in O-PTIR spectroscopy have circumvented this constraint by probing the photothermal effects of IR radiation. Nordholt et al. synthesized fluorescent analogs to the disinfectant active agent, quaternary ammonium compounds (QACs), and elucidated their mode of action through flow cytometry and fluorescent microscopy.In addition to spectroscopic techniques, the collection of articles in this Research Topic contributes to an enhanced understanding of the mechanisms of AMR at the single-cell level. The articles featured in this Research Topic underscore the power and potential of single-cell tools in combating AMR. Many of these exemplify the capability of single-cell techniques to revolutionize clinical microbiology by rapidly and accurately identifying pathogens' type and quantity, and obtaining the antimicrobial susceptibility profile, ultimately resulting in reduced TAT and improved patient outcomes. The showcased advancements in spectroscopic tools, machine learning, and microscopy techniques offer researchers and clinicians powerful tools to study and combat AMR at the single-cell level. We hope that these contributions will inspire further research and innovation in this field, ultimately leading to the development of novel interventions to address the growing threat of AMR.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Since the successful approval of gemtuzumab ozogamicin, antibody-drug conjugates (ADCs) have emerged as a pivotal category of targeted therapies for cancer. Among these ADCs, the use of monomethyl auristatin E (MMAE) as a payload is prevalent in the development of ADC drugs, which has significantly improved overall therapeutic efficacy against various malignancies. However, increasing clinical observations have raised concerns regarding the potential nervous system toxicity associated with MMAE-based ADCs. Specifically, a higher incidence of peripheral neuropathy has been reported in ADCs incorporating MMAE as payloads. Considering the increasing global use of MMAE-based ADCs, it is imperative to provide an inclusive overview of diagnostic and management strategies for this adverse event. In this review, we examine current information and what future research directions are required to better understand and manage this type of clinical challenge.  Health sciences; Medicine; Toxicology; Oncology; Cancer systems biology; Cancer The approval of gemtuzumab ozogamicin in 2000 marked a significant milestone in the development of antibody-drug conjugates (ADCs) as a crucial class of targeted therapies for cancer.,,The MMAE-based ADCs have shown remarkable efficacy in improving overall therapeutic outcomes against various cancers. For instance, brentuximab vedotin treatment in CD30-positive Hodgkin lymphoma and systemic anaplastic large-cell lymphoma patients resulted in overall objective response rates (ORR) of 73% and 86%, respectively.However, increasing clinical observations have raised concerns regarding the potential toxicity to nervous system caused by MMAE-based ADCs, predominantly manifesting as peripheral neuropathy.Considering the increasing global use of MMAE-based ADCs, in this review, we provide an inclusive overview of peripheral neuropathy induced by MMAE-based ADCs. Through comprehensively addressing the clinical aspects, underlying mechanisms, and management strategies, our work aims to facilitate a better understanding of this adverse effect and inform future research efforts to enhance the safety and efficacy of ADC therapy.Peripheral neuropathy is a commonly observed adverse effect of microtubule-targeting agents in clinical trials, with varying frequencies and levels of severity.,,,,The incidence of peripheral neuropathy of any grade in patients treated with brentuximab vedotin was reported to be between 36 and 69 percent, with approximately 4–14 percent of patients experiencing severe peripheral neuropathy.Treatment with tisotumab vedotin was also associated with peripheral neuropathy. In the innovaTV-204 study, peripheral neuropathy treatment-related adverse events occurred in 33% of patients, with 26% having grade one or two events, and 7% having grade 3 events.,In addition to the specific risk factors identified in MMAE-based ADC clinical trials, it is worth noting that there are also general risk factors, previously observed with other anticancer agents, such as age, comorbidities, dose level (single dose and cumulative dose), infusion time, treatment duration, lifestyle factors , and prior treatments with the other anticancer agents, which may increase the risk of peripheral neuropathy.,Drug-induced peripheral neuropathy is a common form of nervous system toxicity, with chemotherapy agents being the primary cause. Chemotherapy drugs known to cause peripheral neuropathy potentially include platinum-based compounds ,,Based on the properties of MMAE-based ADCs, the induced peripheral neuropathy may be theoretically attributed to: (1) the binding of ADCs to target antigens expressed in peripheral nerve cells resulting in the target-dependent uptake of the ADCs; (2) the non-specific uptake of ADCs by peripheral nerve cells or immune cells; (3) the bystander effects of MMAE-based ADCs; (4) the premature MMAE release during circulation due to unexpected cleavage . The dir,,,,Non-specific uptake plays a crucial part in the distribution of ADCs into healthy cells that cause off-target side effects. The hydrophobicity of ADCs may contribute to their non-specific uptake by normal cells.,,,,,The mechanisms for microtubule-targeting drug-induced peripheral neuropathy have been proposed to be related to the damage to the axon terminal, DRG neuron cells, and Schwann cells.,,,,etc. These cytokines trigger neuroinflammation and convey nociceptive information to DRG neurons, leading to the perception of pain Duloxetine: Duloxetine is a novel selective serotonin and norepinephrine reuptake inhibitor with proven efficacy and tolerability in treating various types of chronic pain.(2),Acetyl-L-carnitine (ALC): ALC is a trimethylated amino acid that plays a crucial role in intermediate metabolism and has demonstrated high efficacy and tolerance in the treatment of various peripheral neuropathies.(3)Amifostin: Amifostin is a normal cell protectant that can be used as an adjunct therapy for tumor radiotherapy or chemotherapy.(4)Mecobalamin: also known as methyl vitamin-B12. As the active form of vitamin B12, mecobalamin is easier to enter neuron cells because of the high affinity with nerve cells, which can promote the utilization of folic acid and nucleic acid metabolism.Patients undergoing antitumor therapy tend to prioritize the therapeutic effect over peripheral neuropathy. In order to avoid the interference of anticancer efficacy, dose delay, dose reduction, or dose discontinuation is currently recommended in response to peripheral neuropathy when patients are receiving antitumor therapy. However, if a severe peripheral neuropathy was observed in patients who have completed anticancer therapy, some medications could be implemented to enhance patients' treatment compliance and improve patients' quality of life and prognosis. For example, some studies have suggested that duloxetine, acetyl-In addition, some non-medical treatments were adopted to mitigate peripheral neuropathy to some extent. As an auxiliary method, acupuncture and moxibustion are safe and effective with a low incidence of adverse reactions. In a randomized controlled trial to evaluate the feasibility and safety of acupuncture intervention in patients with moderate to severe peripheral neuropathy with breast cancer, eight weeks of acupuncture intervention compared with conventional care significantly improved peripheral neuropathy neurosensory symptoms in patients with mild and moderate breast cancer, with no serious adverse effects observed.Drawing upon our current knowledge and understanding, we proffer algorithmic recommendations for the diagnosis and management of peripheral neuropathy associated with MMAE-based ADCs, as depicted in Currently, the number of survivors for cancer patients is increasing due to the rapid development of novel anticancer therapies. Although the mechanism of antitumor action of chemotherapy drugs is clear, the mechanism leading to peripheral neuropathy may differ, leading to the unpredictability of peripheral neuropathy occurrence and development. The occurrence, severity, and duration of symptoms vary widely among individuals. To achieve better, comprehensive, and satisfactory efficacy, further in-depth and extensive exploration is necessary, and there is still a long path ahead in this area of research.,One especially attractive route might be the improvement of ADC technology to reduce the incidence and severity of peripheral neuropathy associated with ADC therapy. Traditional ADCs often utilize random conjugation methods, which can result in heterogeneous mixtures with varying drug-to-antibody ratios (DAR). However, site-specific conjugation techniques, such as enzymatic conjugation or site-specific bioconjugation, were recently developed to allow for precise and controlled drug attachment to specific sites on the antibody.Secondly, early detection and prevention of peripheral neuropathy associated with MMAE-based ADC therapy can help mitigate its impact and improve patient outcomes. To this end, a comprehensive, convenient, and effective assessment tool is urgently needed to reflect the actual level of patients to the greatest extent. Peripheral neuropathy can cause peripheral nerve damage in patients, reducing their ability to perform daily activities and their quality of life. And peripheral neuropathy symptoms also affect the patient’s psychological, social, and spiritual aspects. Therefore, an ideal comprehensive assessment tool for peripheral neuropathy should include an assessment of the patient’s symptoms and their impact on quality of life and psychological, spiritual, and social relationships.,Additionally, better medical treatments will be required to improve peripheral neuropathy outcomes. The application of nanotechnology-based drug delivery systems presents a promising approach to overcome the low solubility and the bioavailability of certain compounds to relieve peripheral neuropathy. The utilization of nanocarriers in pain management represents a nascent and burgeoning field of inquiry with substantial prospects for clinical advancement.In conclusion, monitoring and managing peripheral neuropathy in patients receiving MMAE-based ADCs are essential for promoting patient well-being, maintaining treatment efficacy, and optimizing treatment outcomes. A proactive and multidisciplinary approach involving oncologists, neurologists, pain specialists, and supportive care teams is crucial to effectively monitor, assess, and manage peripheral neuropathy in these patients. We expect the establishment of better guidelines for the management and monitoring of peripheral neuropathy induced by MMAE-based ADC in the near future.


Developmental Biology is a broad field where each type of biological data at the molecular, tissue, or phenotype level is pertinent to uncover an organism’s origins and developmental process. The final panorama is that each layer of information requires distinct mindsets for accurate data interpretation.Nothing in biology makes sense except in the light of evolution,”  is one of the most interdisciplinary subareas of Bioinformatics. NSB became an invaluable tool for understanding biological systems, for the formulation of original hypotheses, for comparison between different organisms, and for interpreting a massive amount of data . This poIn silico structural analysis at the protein level is also a common integrative approach, combined with data from the wet lab or as a stand-alone analysis. Analyzing a single protein structure can be considered an entire universe of complexity because numerous investigations need to be applied to infer a molecular behavior or a possible structural feature. In this sense, there is a demand for more computational methods to analyze proteins essential to the developmental process. Due to the numerous limitations of experimental techniques at the wet lab, many proteins, such as HOX proteins, have no complete structural data. Therefore, the precision of protein structural modeling tools, especially using ab initio calculations, significantly increases the success of protein structure research  approaches are gaining territory in almost all biological and Biomedical Sciences. Such tools’ vast applicability can be adapted to virtually all Big-Data problems. Either being applied to expression , genomicThis Research Topic presents some integrative approaches applied to developmental biology experiments from different backgrounds.Fagny et al. employed systems biology to integrate genomic, transcriptomic, and epigenomic data to elucidate the regulatory relationships between transcription factors, enhancers, and potential target genes in maze and identify regulatory key factors specific to leaves at the seedling stage and husks at flowering. By combining different omic data, they reconstructed tissue-specific-associated transcriptomic factors regulatory networks and uncovered genes crucial to tissue-specific differentiation.Zhou et al. combined two RNA-seq datasets of human placentas from term and preterm birth to investigate co-altered circadian transcripts-associated long intergenic non-coding RNAs (lincRNAs). Using a diverse set of transcriptomic analyses, they uncovered nine core molecular clock genes deregulated by the decrease of five circadian lincRNAs in the placenta, affecting a myriad of crucial biological processes that could be linked to preterm birth.Shang et al. analyzed cardiac development-associated transcriptomic data from three Single-Cell Tagged Reverse Transcription (STRT-Seq) datasets. Using a transcriptomic analysis workflow, they explored lineage-specific changes in mouse and humans regarding gene expression, subpopulation composition, and developmental features in cardiac tissues. They described an evolutionary conservation of cell populations and molecular profiles during heart development in both species.Tang et al. reviewed experimental technologies, public data, and predictive models associated with synthetic lethal pairs. The knowledge of synthetic lethal pairs is deeply interconnected to developmental biology since it revolves around how the impairment of two genes can lead to cellular or organism death, which does not happen when one of these genes is still viable. The authors outlined important perspectives and critical discussions on the subject that can aid future research.Wu et al. provided a comprehensive review of the mathematical model of local ancestry inference (LAI) applied to genomic data. In this sense, its application, historical aspects, different benchmarks, and the strengths and limitations of LAI are discussed and outlined.Finally,


Multiple myeloma (MM) is the most common primary bone-originating tumor, whereas extramedullary plasmacytoma (EMP) is a plasma cell tumor that arises outside the bone and is most commonly found in the head and neck area. Gastrointestinal and particularly gallbladder involvement is exceedingly rare, and symptoms, if any are present, are usually similar to those seen with cholelithiasis. Treatment options usually include surgical resection and/or chemotherapy. In this report, we present a rare case of a clinically unexpected plasmablastic extramedullary plasmacytoma that was found on abdominal ultrasound (US) and magnetic resonance imaging (MRI) in a 61-year-old asymptomatic patient and led him to undergo cholecystectomy. A fluorodeoxyglucose positron emission computed tomography (FDG PET-CT) that was performed due to the onset of left thigh pain also demonstrated concurrent bone plasmacytoma. The patient is currently receiving chemotherapy and is also being prepared for autologous stem cell transplantation. In this context, we further present the diagnostic, therapeutic and prognostic challenges of EMPs. Lastly, we point out the distinct features of the plasmablastic subtype and analyze its differences compared to other histologic subtypes in achieving a successful diagnosis and management.