Cocaine addiction is a serious condition with potentially lethal complications and no current pharmacological approaches towards treatment. Perturbations of the mesolimbic dopamine system are crucial to the establishment of cocaine-induced conditioned place preference and reward. As a potent neurotrophic factor modulating the function of dopamine neurons, glial cell line-derived neurotrophic factor (GDNF) acting through its receptor RET on dopamine neurons may provide a novel therapeutic avenue towards psychostimulant addiction. However, current knowledge on endogenous GDNF and RET function after the onset of addiction is scarce. Here, we utilized a conditional knockout approach to reduce the expression of the GDNF receptor tyrosine kinase RET from dopamine neurons in the ventral tegmental area (VTA) after the onset of cocaine-induced conditioned place preference. Similarly, after establishing cocaine-induced conditioned place preference, we studied the effect of conditionally reducing GDNF in the ventral striatum nucleus accumbens (NAc), the target of mesolimbic dopaminergic innervation. We find that the reduction of RET within the VTA hastens cocaine-induced conditioned place preference extinction and reduces reinstatement, while the reduction of GDNF within the NAc does the opposite: prolongs cocaine-induced conditioned place preference and increases preference during reinstatement. In addition, the brain-derived neurotrophic factor (BDNF) was increased and key dopamine-related genes were reduced in the GDNF cKO mutant animals after cocaine administration. Thus, RET antagonism in the VTA coupled with intact or enhanced accumbal GDNF function may provide a new approach towards cocaine addiction treatment. Cocaine is one of the most commonly consumed drugs of abuse worldwide  with potConditioned place preference (CPP) is an important test for assessing the rewarding effects of drugs of abuse ,10,11. CN-terminal kinase (JNK) pathway [Glial cell line-derived neurotrophic factor (GDNF) is potentially the strongest dopamine function promoting secreted neurotrophic factor ,21,22. I pathway ,41.Mice with constitutively active RET elicited via mimicking the Met918Thr mutation from multiple endocrine neoplasia type B (MEN2B) ,43 displTaken together, current data suggest that GDNF-GFRa1-RET signaling is an important modulator of addiction to commonly used drugs of abuse and may therefore prove to be a viable therapeutic approach to addiction treatment . In lineMouse husbandry was performed as described previously . BrieflyGenotyping was performed as described previously . Genotyp14 VG/mL. Intrastriatal AAV5-cre injections were performed as described previously . BrieflyOnce the injection was completed, the skin above the skull was sutured closed and the animals were administered carprofen (5 mg/kg)  as an analgesic. The next day, sutures were checked to ensure proper wound closure and healing. In the following days, the animals were monitored to ensure proper recovery, and kept for at least 2 days prior to beginning experiments.Conditioned place preference procedures were carried out as described previously ,44,50. CConditioned place preference was then evaluated the following day with the mice allowed to explore both compartments for 15 min. Preference was scored as the difference between time spent on the cocaine-paired side minus the group average time mice spent on the saline-paired side to reduce bias. After preference was established, mice were intracranially injected with AAV2/5-Cre developed previously  over 2 d2 and/or cervically dislocated, followed immediately by decapitation. The skull was quickly cut open and the brain removed and placed into ice-cold PBS prior to tissue isolation. Brain structures of interest were then dissected from 2 mm thick slices using a brain matrix (Stoetling) and immediately snap frozen using dry ice and stored at −80 °C until use for RNA extraction.Animals were deeply anaesthetized with COq value for one or more of the sample duplicates was 40 or 0, or when the Cq difference between replicates was >1. Primer sequences used are depicted in Reverse transcription and quantitative PCR (qPCR) was performed as described previously . Brieflyt-tests with Welch’s correction. Gene expression changes relative to wild type where multiple genes, and thus multiple hypotheses, were tested at once were determined using multiple t-tests with a single, pooled variance followed by multiple comparison correction by controlling for the false discovery rate with the Benjamini, Krieger, and Yekutieli two-stage set-up method such that a rate of 5% (Q = 5) was accepted for determining significance.All statistical analyses were performed using GraphPad prism software. Any potential outliers were identified by Grubbs’ test with an alpha = 0.05 and removed prior to subsequent analysis. Behavioral data with repeated measures was analyzed using 2-way repeated measured ANOVA, or mixed-effects analysis if values were missing, followed by post-hoc Holm–Šídák tests to ascertain significant effects. Gene expression changes at the mRNA level and non-repeated behavioral data were analyzed using Clinical management of cocaine use disorder requires interventions after cocaine addiction has previously been established. To this end, cocaine-induced conditioned place preference was first established prior to reducing RET expression in the mesolimbic dopamine pathway of RET conditional knockout (cKO) mice  via intrp < 0.05; t-test p < 0.05; p = 0.16).No significant differences in overall locomotor activity were observed between the genotypes during CPP testing, with differences only occurring as an effect of saline or cocaine treatment A. Notabla priori between the genotypes before AAV-Cre injections were performed (a priori preference difference could bias results and make interpretation of any effects due to reduction of GDNF difficult, group 1 was excluded from further analysis of CPP. Group 2 did not have significant preference differences observed a priori between the genotypes (The effects of GDNF on dopamine neurons are mediated by RET ,35. Howeerformed D. Becausenotypes E, and thPrior to AAV-Cre injections, animals of both groups displayed decreased locomotor activity after saline injections and increased locomotor activity due to cocaine, and, as expected, no differences between genotypes were observed A. DuringAfter AAV-Cre injection, the mutant animals of group 2 showed markedly prolonged CPP extinction, with preference failing to reach control levels, even after 1 month B. ReinstNext, we addressed how NAc GDNF affects accumbal dopamine function by analyzing the expression level of dopamine-related and plasticity genes. We found that within the NAc, mRNA expression of proteins associated with dopamine biosynthesis and function, DAT, TH, DRD1, DRD2, and VMAT2, were largely unchanged, as were Ret and Gfra1 mRNA levels A. HoweveConstitutively active RET has been shown to strongly prolong CPP extinction to amphetamine , suggestt-test, indicating these groups are not inherently different. Future studies are required to determine whether constitutive adult-onset antagonism of RET would also be beneficial, or if it is specifically the reduction of RET levels in the dopamine neuron cell bodies in the VTA which has potential clinical benefit.We report that a 60–70% reduction of RET after the onset of cocaine addiction in the VTA hastens CPP extinction and reduces preference upon reinstatement compared to controls. Thus, partial inhibition of RET in the VTA may be beneficial in treating cocaine addiction. However, here we have targeted only the VTA for RET reduction. It is interesting to note that the difference between habituation and cocaine locomotor activity prior to AAV injection was not significant in the RET wt/wt animals, whereas it was significantly different in the RET cKO/cKO animals. This could indicate an initial difference in response to cocaine already inherent in the animals prior to RET reduction. However, it is most likely that this lack of significant effect in the wild type animals is due to the higher variation in the wt group. Importantly, there was no significant difference in locomotor activity observed between the RET wt/wt animals and the RET cKO/cKO animals when analyzed by Importantly, we found that endogenous RET reduction in the VTA and endogenous GDNF reduction in the NAc have opposite effects on CPP extinction and reinstatement. While it is unlikely due to the low expression of neurturin or GFRa2 in the NAc ,56, neurIt is interesting that differences in behavioral response to cocaine administration were not observed between genotypes, as locomotor activity was not significantly different between genotypes. This suggests a specific role of NAc GDNF and VTA RET signaling in cocaine addiction, but not cocaine-induced motor activity. Indeed, there is a nonsignificant trend towards an increase in locomotor activity in the RET cKO animals with about 70% reduction of RET levels during reinstatement despite having reduced cocaine preference. Together, this could indicate the possibility of a specific role of GDNF-RET signaling in the cocaine reward.We also observed an upregulation of BDNF in the NAc of GDNF cKO animals, which is potentially of particular relevance to the observed prolonged CPP extinction. Namely, BDNF and TrkB knockdown in mouse NAc has been shown to reduce CPP ,62,63,64We also found that after cocaine administration, a 70–80% reduction of GDNF in the NAc moderately reduces DAT-, RET-, and TH-encoding mRNA expression in the VTA. The reduction in dopamine-related gene expression could also be due to histological alterations in midbrain dopamine neurons such as fiber or cell degeneration. However, degeneration is unlikely as GDNF has been demonstrated to not be required for the survival of midbrain dopaminergic neurons in vivo in GDNF knockout animals . Still, GDNF itself has for decades been known to have potent effects on dopamine system function and survival in models of Parkinson’s disease ,21. NotaIt is intriguing that a reduction of GDNF levels in the NAc of GDNF cKO animals also resulted in a modest reduction in RET mRNA levels in the VTA. Given that the reduction in RET mRNA levels in the GDNF cKO is associated with prolonged CPP, whereas the reduction of RET alone in the VTA hastened CPP extinction, this could indicate that a reduction of RET expression may not have a significant or specific role in decreasing cocaine-induced CPP, or, alternatively, that maintaining or amplifying NAc GDNF is necessary in addition to reducing VTA RET expression to hasten cocaine-induced CPP extinction. It is interesting to note the potential nonsignificant trend towards increased GDNF in the VTA of the RET cKO animals. This indicates support for the latter hypothesis. However, future studies are required to adequately resolve this interesting question.Clinically, pharmacotherapeutic management of cocaine addiction would require treatment to be applied after cocaine use disorder has already been established to attain an initial period of abstinence and assist in preventing relapse . Here, w


In the original publication , there wa correction has been made to *4. Discussion, Paragraph 4*:With regard to the correction of **The results showed that formaldehyde treatment reduced the homogeneity of virus particles. Although there were no significant differences in the negative staining or circular dichroism spectra, the treament’s performance in terms of size exclusion chromatography became worse, and its UV absorption curve became less sharp. An examination of the S protein showed that the viral S protein after formaldehyde treatment displayed dispersive bands and cross-linking upon SDS-PAGE. Compared with the 20–24 h BPL treatment, the S antigen content decreased by more than twice. After BPL treatment, the virus particles remained relatively homogenous, and the S protein was still clear. These results confirm the chemical properties of formaldehyde, which can cause viral protein cross-linking and denaturation and damage viral surface antigens. Therefore, although formaldehyde does not destroy the morphology or secondary structure of the virus, compared with BPL, formaldehyde is not a suitable SARS-CoV-2 inactivator.**


Fetal cardiac MRI is a rapidly evolving form of diagnostic testing with utility as a complementary imaging modality for the diagnosis of congenital heart disease and assessment of the fetal cardiovascular system. Previous technical limitations without cardiac gating for the fetal heart rate has been overcome with recent technology. There is potential utility of fetal electrocardiography for direct cardiac gating. In addition to anatomic assessment, innovative technology has allowed for assessment of blood flow, 3D datasets, and 4D flow, providing important insight into fetal cardiovascular physiology. Despite remaining technical barriers, with increased use of fCMR worldwide, it will become an important clinical tool to improve the prenatal care of fetuses with CHD. While fetal echocardiography remains the first line to diagnose cardiovascular anomalies in-utero, MRI has increasingly been used as a complement to ultrasound-based modalities. Fetal echocardiography was first introduced in the mid-1980s . There aIn contrast to other organ systems, assessment of the fetal cardiovascular system requires dynamic imaging to resolve cardiac motion and blood flow. Fetal cardiac MRI (fCMR) to date has been technically limited due to fetal motion, small size of fetal structures, and lack of a real time fetal electrocardiogram (ECG) to synchronize data acquisition to the fetal cardiac cycle. Innovative technology has allowed some of these barriers to be overcome and increased the potential for application of fCMR clinically . In addiThis paper will review the state-of-the-art of fCMR, including new technologies instituted in recent years, methods and workflow of fCMR, safety, current clinical applications of fetal MRI and fCMR, and opportunities for innovation.There are inherent challenges of motion, cardiac gating, and achieving adequate spatiotemporal resolution that have limited the clinical use of fCMR . CardiacWhile fetal electrocardiogram devices have been available for a century as research tools, electrocardiography in the fetus has not yet become a part of routine clinical care. Prior work has required intensive signal averaging  or had sfCMR has been successfully performed using both 1.5 and 3 T MR scanners . Fetal CThe most common sequences utilized for static imaging of the fetal heart include balanced steady-state free precession (bSSFP) and single-shot fast spin echo or “black blood” sequences including half-fourier acquisition single-shot turbo spin echo (HASTE) and single shot fast spin-echo (SS-FSE). b-SSFP allows for contrast between the blood pool (hyperintense) and myocardium (hypointense). The fast spin echo sequences produce T2 weighted images with black blood contrast that provide excellent contrast between the vasculature and lung/thymus . The benTime resolved (CINE) bSSFP imaging can be utilized with gating to obtain dynamic high spatial and high temporal resolution images  that provide an assessment of both cardiac structure and function. Acquisition time of each slice of gated bSSFP cine imaging is a few seconds, making it susceptible to both fetal motion and maternal breathing .To minimize artifacts from maternal respiration, maternal breath holds or shallow maternal breathing has been utilized with success . HoweverThere is no sedation or contrast used for fetal CMR. After a scout acquisition, a series of static images in multiple planes  are obtained. Dynamic images using gating with cine b-SSFP sequences in multiple planes  are obtained for each fetus. Multi-slice imaging to provide whole heart coverage using parallel overlapping slices in three orthogonal planes or single-slice imaging in specific imaging planes of interest can be utilized.The typical scanning time studies have reported are 30–60 min; however, placement of the DUS device and finding a stable fetal heart rate signal often adds time to the scan. CMR localizer sequences or fetal echocardiogram images can be used prior to the scan to help visualize the position of the fetus and fetal heart prior to DUS device placement. Some centers have successfully utilized imaging post processing techniques to produce higher resolution images .Phase-contrast MRI (PC-MRI) is a powerful tool that has allowed for non-invasive quantification of blood flow and is the gold standard for the hemodynamic assessment postnatally in children with congenital heart disease  With theBeyond 3D anatomy and cine functional assessment, cardiac MRI allows for three-directional velocity encoding (4D flow) to comprehensively analyze vascular and valvar flow in the fetal heart. 4D flow provides qualitative dynamic blood flow imaging by streamline mapping or particle tracing to visualize phasic flows such as ductal shunts, atrial shunts, and venous flows , 22. TheAt the same time, considerable technical challenges remain, accounting for a considerable failure rate (∼25%) in feasibility studies . 4D flowin-vivo investigations of flow characteristics governing heart remodeling/disease progression that were previously limited to computational methodologies  or restriction of the foramen ovale in hypoplastic left heart syndrome (HLHS) . PulmonaPostnatal cardiac MRI is a common component of the pre-operative planning for many surgical procedures in the neonate. Similarly, subtleties in the cardiac anatomy can have major prognostic significance which may be useful in counseling prenatally. At present, the use of fCMR clinically is in its infancy. fCMR can clarify anatomy and physiology across a wide range of CHD. Prior studies have shown feasibility of performing fCMR in specific clinical scenarios including ectopia cordis , fetal ain utero. fCMR has been used successfully to quantitatively assess ventricular volumes, showing agreement with fetal echocardiography  extracardiac vascular structures, including aortic arch shape , 2) sys sys2), afCMR is a promising technology that can be used in conjunction with fetal echocardiography to improve accuracy of diagnosis, heighten sophistication of counseling and postnatal planning of CHD, and provide a comprehensive assessment of the cardiovascular system. Additionally, it may help cardiovascular surgeons and interventionalists plan for neonatal care in the fetal period. Data to support clinical insurance coverage for indications for fCMR are yet to be defined, though preliminary work suggests a role in both extracardiac vascular examination and intracardiac ventricular size and function. Research with phase contrast imaging, 4D flow, and T1 and T2 mapping may allow for a deeper understanding of fetal physiology including oxygen delivery and consumption in different forms of CHD and heart failure.New technology, including DUS device enabling fetal cardiac gating, has allowed more centers to start performing fCMR. There are inherent technical challenges including achieving adequate spatiotemporal resolution and availability of trained staff and physicians to acquire, interpret, and communicate the imaging findings to the care team. Research in fetal electrocardiography may eventually allow for direct fetal cardiac gating, similar to postnatal CMR. With new technology in this rapidly advancing field, image, flow, and volume acquisition will be easier and more centers will obtain experience performing fCMR, allowing for it to be a more accessible and useful imaging tool in clinical care. Future research will help to delineate the clinical use of fCMR and effect on fCMR on long term outcomes of prenatally diagnosed CHD.


In recent years, 3D printing (3DP) has advanced traditional medical treatments. This review explores the fusion of reverse engineering and 3D printing of medical implants, with a specific focus on drug delivery applications. The potential for 3D printing technology to create patient-specific implants and intricate anatomical models is discussed, along with its ability to address challenges in medical treatment. The article summarizes the current landscape, challenges, benefits, and emerging trends of using 3D-printed formulations for medical implantation and drug delivery purposes. Acquisition of 3D geometrical data: This initial phase involves digitally capturing 3D geometric information. These data can be directly gleaned from the patient or extracted from their medical records. Advanced imaging techniques such as computed tomography (CT) or magnetic resonance imaging (MRI) play a pivotal role in this stage, precisely capturing patient-specific anatomical details.Modification and Adaptation: Following data acquisition, the subsequent step entails modification and adaptation procedures. This stage serves as a bridge between raw data and tailored solutions. It involves fine-tuning the acquired data to align with the unique requirements specific to the patient.Creation of a 3D model or final product: With refined data, the process advances to create a 3D model or the ultimate product. Employing cutting-edge 3D printing technology, these models or products are realized. This stage further encompasses meticulous control over various aspects, including material attributes, shape, dimensions, and, most importantly, patient comfort.Reverse engineering is a fast and cost-effective technique for crafting functional or nonfunctional replicas of existing objects. In the context of rehabilitation, the process of reverse engineering can be delineated into a sequence of coherent stages facilitated through semi-automated means:The confluence of these stages generates a streamlined and efficient process, enabling the realization of patient-specific solutions in targeted drug delivery options. This approach, reinforced by reverse engineering principles, showcases the potential to reshape the redesign of personalized medical interventions for more effective drug delivery for localized regions in cases of chemotherapeutic drugs or drugs with severe side effects.Originating in the 1980s from the automotive and aerospace sectors, 3DP has experienced a transformative journey. While its roots lie in these industries, recent strides in the medical domain have ushered in a wave of revolutionary possibilities, particularly within the pharmaceutical sector, where the concept of personalized medicine has found fertile ground for growth [Three modalities of 3DP have been explored for producing pharmaceutical formulations: laser-based writing systems, printing-based inkjet systems, and nozzle-based deposition systems . Laser-bPolyjet or multijet printing (MJP) offers flexibility in producing models of varying density and color, but the main disadvantage is the high cost . The firThe first FDA-approved oral drug delivery system, “Spritam ,5” was dA high dose cardiometabolic drug demonstrated success, disintegrating in seconds with positive stability and bioavailability. Aprecia later shifted its focus to central nervous system drugs, particularly levetiracetam, leading to the approval of SPRITAM, marking the first regulatory approval for a pharmaceutical product manufactured using 3D printing. The approval of SPRITAM also signifies a renewed interest in applying 3D printing and other additive manufacturing techniques to pharmaceuticals and life sciences over the last decade.Diverse filament options: FDM offers a broad selection of filament materials providing flexibility in design and functionality.Speed and efficiency: This technology allows for rapid production ensuring a quick turnaround time for finished parts and assemblies.Cost-effectiveness: It is more affordable compared to other 3D printing techniques, making it budget-friendly choice for various applications.Durability: It produces robust and sturdy models, making it ideal for parts intended for harsh environments or heavy use.Complex geometries: It excels in handling projects involving large pieces and intricate designs, often at a lower cost to size ratio than alternative methods.Precision and consistency: This method enables high precision production with consistent and repeatable results, ensuring quality across multiple iterations.Toolless manufacturing: All that is required to create a part is a 3D printer, eliminating the need for specialized tools or molds.Environmentally friendly: This technique is eco-friendly emphasizing sustainable practices in manufacturing processes.Among the various other methods of 3D printing, FDM stands out as the most popular. This technique utilizes heat to liquefy a filament made of FDA-approved thermostatic material PLA. The molten filament is guided through an extruder and meticulously deposited layer-by-layer to create three-dimensional prints. FDM 3D printing proves to be an exceptional choice for producing functional prototypes and end-use parts due to its capability for precision and versatility. FDM has gained popularity due to some advantages over other printing methods. Some of the benefits include:Despite its numerous advantages, FDM does has some drawbacks. One notable limitation is that FDM-printed parts tend to exhibit lower resolution and rougher surface finishes when compared to those crafted through techniques such as SLA and some other 3D printing methods.3DP works similarly as various functional materials are deposited on polymeric substrates, representing an intersection between extrusion-based printers and laser-based systems. UV light is used to harden the polymer after it is extruded onto the substrate . FurtherAdditive manufacturing techniques enable customizability, especially in the production of personalized medicines with precise dosages allowing high control over release profiles and delivery locations. Commonly used 3DP techniques used for drug delivery applications include FDM, SLA, powder bed fusion (PBF), and inkjet printing.Fused deposition modeling is a commonly used 3DP technique in the pharmaceutical and medical sectors. In FDM, a thermoplastic filament is melted and extruded layer-by-layer to create the desired structure. FDM offers several advantages for drug delivery applications, such as the ability to create complex geometries, control the porosity of the structure, and incorporate drugs into the filaments.One of the most popular applications of FDM in drug delivery is the creation of drug-loaded filaments. Pharmaceuticals are first embedded into the polymer matrix and extruded as filaments of the required dimensions, which are then used in the 3DP machine to create a prototype or drug delivery device that can be orally used as tablets, implanted, or injected into the body. The drug concentration and release can be controlled by adjusting the amount of drug to be loaded in the polymer matrix as well as changing the porosity of the model that will be 3D printed.Scoutaris et al. 3D-printed indomethacin-loaded chewable tablets using polyethylene glycol (PEG) polymer filaments using the FDM technique. For enhanced acceptability of the tablets by pediatric patients, formulations were fabricated in the form of variable shapes, including a lion, heart, bottle, ring, and bear . Sadia eStereolithography: SLA is a 3DP technique that uses a laser to solidify a liquid resin layer-by-layer to create a 3D structure. SLA has emerged as a promising technique for drug delivery applications due to its ability to create complex and precise structures with high resolution. Xu et al. formulated ibuprofen-loaded mini-sized pellets for oral administrative applications using SLA technology. They have used polyethylene glycol diacrylate (PEGDA) as monomer and diphenyl  phosphine oxide (TPO) as photo initiator . In a reSLA has also been used to create microneedle arrays for transdermal drug delivery. Economidou et al. used SLA to create microneedle arrays containing insulin in both pyramidal and spear-shaped microneedles for transdermal administration. The evaluation of these 3D-printed microneedles in a mice model showed an enhanced skin penetration compared to subcutaneous injections . Xu et aIn the PBF type of 3DP, a layer of powder is spread across the build plate and selectively melted using a laser or electron beam to create the desired shape. Additive manufacturing techniques including selective laser sintering (SLS), selective laser melting (SLM), and electron beam melting (EBM) are some of the common methods that can be categorized under PBF. Of all these techniques, SLS is the only non-metallic process that can utilize biocompatible and biodegradable material to produce 3D-printed objects. This process has a close resemblance to traditional tablet-manufacturing process. Additionally, this technique enables creating complex and porous structures with high resolutions. Due to these advantages, researchers have explored SLS technology and its assisted materials, for numerous drug delivery applications .Gueche et al. utilized SLS type of PBF technique to manufacture solid oral dosage forms from copovidone and paracetamol using carbon dioxide laser sintering. Authors in this research have demonstrated that utilization of copovidone has allowed fabrication of dosage forms without additional absorbance enhancers . In anotIn inkjet printing, droplets of drug-loaded resin are ejected/sprayed on the substrate/build platform in layer-by-layer fashion fusing with layers underneath to create an object of the desired shape. Inkjet printing has demonstrated great potential for drug delivery applications, particularly for creating precise and personalized drug delivery systems. Its ability to create complex patterns with high resolution makes it an attractive option for developing advanced drug delivery devices.In a research study conducted by Boehm, anhydride copolymer microneedles were manufactured using SLA-type 3DP technology, where these needles were surface-coated with miconazole using inkjet technology to produce transdermal microneedles for cutaneous fungal infection treatment . SimilarDrug delivery systems are printed with different polymers and by varying methods depending on the goal of the print . PolymerHowever, these compounds have the drawback of being less biocompatible . The secMost synthetic biopolymers such as PLA, PGA, PLGA, and PCL are 3D-printed using the FDM technique. This method requires the polymers to be heated to their melt extrusion temperatures. These temperatures are higher than the melting temperature. Depending on the composition of the polymer, the melt extrusion temperatures range from 90–220 °C. Viscoelastic properties of these polymers are highly dependent on temperature and composition. Drugs or bioactive agents that are thermally stable can be used with these polymers. These polymers are also used in SLS-based 3DP; fine powders of the polymer are melted using high energy lasers .The other biomaterials used in extrusion or FDM printing include synthetics  and natural polymers . These biomaterials undergo crosslinking when exposed to suitable external stimuli. The crosslinking stimuli can be physical  and chemical (counter ions) stimuli. The mechanical properties of the biomaterials improve after crosslinking as polymers are held in place by covalent and ionic bonds. Mechanical properties of biomaterials are very important as they determine how the material maintains shape, retains architecture, and enables easy handling of the 3D-printed structures .Another important property of biomaterials that needs to be considered is the degradation process, products of degradation and route of elimination. Polymers primarily undergo bulk erosion or molecular degradation. In the case of bulk erosion, the scaffolds undergo hydrolysis at random ester bonds and undergo further hydrolysis to release monomers into the tissue. Depending on the type of polymer used, these monomers could be lactic acid, glycolic acid, fumarate etc. These monomers, components of physiological processes such as the Krebs cycle, are eliminated through the lungs . BioceraBiomaterials used in vat polymerization-based 3DP methods such as SLA and DLP are modified by addition of acrylate groups. Commonly used biomaterials include gelatin methacrylate (GelMA), polyethylene glycol diacrylate (PEGDA), and hyaluronic acid methacrylate (HAMA), etc. Methacrylate and diacrylate polymers in the presence of photointitators such as Irgacure, LAP, etc. undergo free radical polymerization when exposed to UV or visible light to enable crosslinking. These photoinitiators are cytotoxic and hence should be used with caution. The biomaterials used in vat polymerization methods have lower viscosity on the millipascal-second order. The resolution of the objects printed using vat polymerization depends on the energy and point size of the light source .In binder jet-based 3DP, two biomaterials are used—one in the powder form and the other in the liquid form. The packing density, particle size and flowability of the powder are important properties. Printability and resolution of the printed objects depends on the size of the powder particles. Layer thickness in binder jet printing is higher than the particle size (of the powder) and ranges from 15–300 μm. A few of the commonly used biomaterials in binder jet 3DP include titanium and its alloys, cobalt–chromium alloys, calcium phosphate salts, polymers, and composites. The binders provide mechanical stability to the printed object by gluing the powder particles together. Low-viscosity materials such as PVA solution, water, phosphoric acid, etc., are used as binders for biomedical applications .Drug delivery via implants can be a very effective manner of drug delivery, benefitting those patients who need long-term treatments. The aim of using reverse-engineered 3DP implants is to create a patient-specific, reproducible loading for effective doses of drugs by using MRI/CT scans. Loading can be accomplished via incorporation into the printing process or during the printing process itself. When using FDM printing, drugs can be incorporated in making the filaments as seen in The drugs are coated to the pellets using oil casting ,34,35. TEscherichia coli (E. coli) and a decrease in number of osteosarcoma cells respectively. The process of loading the filament with drugs and printing the 3D disc did not reduce the effectiveness of the drugs. After post-processing, Boyer et al. [Sandler et al. , showed r et al.  showed tr et al.  and Boyer et al. .3D-printed catheters embedded with antibiotics and chemotherapeutics using FDM technology were shown to have an initial burst of release followed by a steady release rate for five days by Weisman et al. . Figure One current example of drug-releasing devices is the intrauterine device (IUD), which provides long-term contraception with localized hormone delivery . One issMaterials such as flexible TPU have been used to print bacteriostatic vaginal meshes. These meshes were loaded with doses of levofloxacin for treating stress urinary inconsistence and pelvic organ prolapse . Zhao etS. aureus and S. epidermidis.Research has also shown that drugs such as minocycline, gentamicin, isoniazid, rifampicin, and vancomycin, when used in 3DP implants, have been helpful in treating bone fractures and other injuries ,50,51,52Surgical meshes are classically used to treat hernias . 3DP tecUtilizing reverse-engineered three-dimensional printing (3DP) technology for the localized delivery of antibacterial agents at the implant site presents significant potential in mitigating infection and promoting implant longevity throughout the recovery period. In the context of cancer treatment, numerous antitumor medications exhibit low solubility in aqueous solutions . CurrentInfuse Bone Graft: This implantable device, manufactured by Medtronic, delivers recombinant human bone morphogenetic protein-2 (rhBMP-2) to promote bone growth in spinal fusion procedures. 3D printing could create patient-specific implants with optimized geometry and drug release profiles, improving surgical outcomes and reducing complications.Zoladex: A biodegradable implant developed by AstraZeneca for treating prostate cancer and certain gynecological disorders. It releases the drug goserelin over time, which helps regulate hormone levels. 3D printing could enable the development of implants with customizable drug release rates and more precise control over hormone regulation.Norplant: A subdermal contraceptive implant that releases the hormone levonorgestrel over an extended period. It has been replaced by newer systems such as Nexplanon and Implanon. 3D printing could be used to develop patient-specific implants that deliver the optimal drug dose based on individual patient needs and characteristics, potentially reducing side effects and improving efficacy.Vitrasert: An ocular implant used to treat cytomegalovirus retinitis in patients with AIDS. The implant releases the antiviral drug ganciclovir over an extended period. 3D printing could be used to develop customized ocular implants that conform better to individual patient anatomy, improving drug delivery and reducing complications.Probuphine is a subdermal implant that delivers buprenorphine to treat opioid dependence. Titan Pharmaceuticals developed the implant, which provides continuous drug release for up to six months. 3D printing could create personalized implants that optimize drug release based on individual patient needs, potentially improving treatment outcomes, and reducing relapse rates.Implantable drug delivery devices: Several commercially available implantable drug delivery devices have been developed to provide controlled drug release over an extended period. Some examples include:Customization: 3D printing enables the creation of patient-specific implants that match individual patient anatomy and clinical needs, potentially improving treatment outcomes and reducing complications.Precision: 3D printing allows for precise control over implant geometry, material properties, and drug release profiles, which could lead to more effective and safer drug delivery.Complex geometries and multi-component systems: 3D printing can produce implants with intricate structures and multiple drugs, allowing for more sophisticated drug delivery strategies.Rapid prototyping and production: 3D printing technologies enable faster development and production of implantable drug delivery devices, potentially speeding up bringing new devices to market.3D printing technologies can potentially improve upon these implantable drug delivery systems by offering:Drug release rate refers to the speed at which drugs become pharmacologically active . The maiA sustained release drug-delivery system consistently delivers a drug overtime to overcome rapid metabolization or elimination by the body . Giri etResearch in developing 3DP is on the way to more promising improvements in the medical sector. Currently, many challenges are limiting the use of this application such as biocompatible material, quality control, and regulatory acceptance. There are many 3DP technologies available, which generates a need to test and compare the different parameters and optimize the drug dosage with efficacy and stability. The major drawback in employing 3DP technologies is the use of UV radiation sources or high temperatures during the 3DP process which can lead to degradation of the embedded drugs. These technologies also require a post-processing step to retain the mechanical properties. Some steps include removal of support, washing of excessive resin and then curing (in the case of SLA and DLP printing), drying (in the case of inkjet printing), and heat step (in the case of powder-based printing). The use of reverse 3DP may make the delivery patient-specific but not necessarily appealing to the patient. The final product may have uneven or rough surfaces. Although 3DP has found extensive use in the medical field, this technology for delivering drugs is still in development, with most of the current research limited to in-vitro studies. Additional studies will be required. There are FDA-approved guidelines for using 3DP medical devices, but there is an absence of official guidelines for dosage delivery. Considering the need for patient-specific manufacturing, regulated specifications should be established for quality control. These are important considerations before 3DP can become viable for mass production and usage.Despite its potential, 3DP confronts several challenges to mainstream drug delivery methods. The technology’s capabilities are not entirely on par with traditional conventional approaches. As we peer into the future, particular areas of concern emerge, primarily centered around the resolution and printing speed. The impending evolution must strive to balance minimal energy consumption and low production costs, thereby enhancing the economic viability of this technology. At a micro- and nanoscale level the clarity and precision of prints necessitate marked improvement. Developing novel materials exhibiting high biocompatibility over extended durations becomes critical. In addition, a notable aspiration for the field is the effective bio-printing of tissues and organs, ensuring in vivo functionality that can seamlessly integrate with the living systems they are intended for.These challenges, while formidable, present the impetus for ongoing research and innovation in the reverse 3D-printing arena. Addressing these concerns would pave the way for this technology to find its place alongside and surpass traditional approaches in drug delivery and medical advancement.In recent decades, healthcare has witnessed a remarkable transformation with the emergence of reverse engineering 3D printing. This innovative technology has demonstrated its potential to surmount the limitations encountered by traditional medical treatments. With more than a decade of dedicated research and exploration, 3DP has garnered substantial attention for its ability to address the challenges in medical treatment by creating patient-specific implants, advanced medical devices, and intricate anatomical models. One particularly intriguing avenue of investigation within this field is the reverse engineering of 3D-printed formulations, a process that holds promise for many medical applications.This review article aims to delve into the current landscape of reverse engineering within 3D printing technology, specifically in medical applications for drug delivery. By examining the convergence of cutting-edge 3DP technologies and the demands of the healthcare sector, we endeavor to shed light on the potential benefits, challenges, and emerging trends in developing 3D-printed formulations for medical purposes. Through this exploration, we seek to contribute to a comprehensive understanding of the transformative impact that 3D printing holds for the future of healthcare.An emerging frontier, reverse-engineered 3DP introduces an inventive approach to drug delivery, harnessing the potential of tailored dosing strategies and bespoke anatomical conformity. By propelling this field forward, further breakthroughs promise to substantially enhance treatment efficacy, bolster patient adherence, and ultimately advance overall health outcomes.As 3D printing progresses, its role in medicine is poised to expand significantly. Envisioned as a cornerstone of the future healthcare landscape, it is set to lay the foundation for ingenious, patient-centered, and economically viable medical solutions.


Cognitive symptoms are common during and following episodes of depression. Little is known about the persistence of self-reported and performance-based cognition with depression and functional outcomes.N = 623). Participants completed app-based self-reported and performance-based cognitive function assessments alongside validated measures of depression, functional disability, and self-esteem every 3 months. Participants were followed-up for a maximum of 2-years. Multilevel hierarchically nested modelling was employed to explore between- and within-participant variation over time to identify whether persistent cognitive difficulties are related to levels of depression and functional impairment during follow-up.This is a secondary analysis of a prospective naturalistic observational clinical cohort study of individuals with recurrent major depressive disorder . Increasing persistence of self-reported cognitive difficulty was associated with higher levels of depression and functional impairment throughout the follow-up. In comparison to low persistence of objective cognitive difficulty (<25% of timepoints), those with high persistence (>75% of timepoints) reported significantly higher levels of depression  and functional impairment  over time. Examination of the individual cognitive modules shows that persistently impaired executive function is associated with worse functioning, and poor processing speed is particularly important for worsened depressive symptoms.508 individuals (81.5%) provided data (mean age: 46.6, We replicated previous findings of greater persistence of cognitive difficulty with increasing severity of depression and further demonstrate that these cognitive difficulties are associated with pervasive functional disability. Difficulties with cognition may be an indicator and target for further treatment input. Cognitive impairments in major depressive disorder (MDD) include deficits in working memory, attention, executive function, and processing speed, which potentially contribute to low mood, anhedonia, and psychomotor retardation  collected self-reported cognitive difficulty, and was administered every 6-weeks via the THINC-it® smartphone app. The PDQ-5 asks respondents to rate how often during the past 7 days they have experienced difficulties with organisation, concentration, and forgetfulness on a scale from 0 (‘Never’) to 4 (‘Often’). The PDQ-5 has been shown to have good reliability  below healthy control standardized means from healthy controls can indicate cognitive difficulties  were calculated by creating: (i) whether the individual scored ⩾1 s.d. below standardized means reported in a healthy population  quantiles from these percentages resulting in mutually exclusive subcategories for the PDQ-5 and each objective cognitive domain . Participants needed to have completed the relevant assessment at least twice throughout the duration of follow-up for persistence to be included in the analysis.Scores on each of the 30 items of the Inventory of Depressive Symptomatology-Self Report (IDS-SR) measure  . First, we tested whether there were systematic differences between those providing and not providing data for analysis using logistic regression. Cross sectional associations between subjective and objective measures of cognitive difficulty were explored using Spearman's correlational analysis. Associations between the cognitive difficulty persistence and time-varying depression or functioning including individual functioning domains were examined using multilevel longitudinal models, pooling data across all 9 timepoints . Multilevel models handle hierarchically nested data and can account for between- and within- participant variation over time and missing data . The median duration of participation was 539 days (IQR: 407.5–730). There was no apparent association between the total number of THINC-it® assessments and depression severity  or functional ability . A full description of the sample, recruitment and retention rates are available in Matcham et al. , people with increasing persistence of cognitive difficulties reported higher levels of depression and functional impairment throughout the course of follow-up. For objectively-measured cognitive difficulties, when comparing different persistence centiles with the reference group (<25% of timepoints), only the comparison between the most and least persistent group was significant with those with difficulties over >75% of timepoints having significantly higher levels of depression and functional impairment throughout follow-up.® modules highlighted the elements of cognitive performance which consistently impact depression and function outcomes. Highly persistent problems with attention, working memory and processing speed were associated with increased levels of depression and functional impairment throughout follow-up. The largest effect sizes were seen in associations between executive function and depression and functional outcomes. Those with highly persistent (>75% of timepoints) problems with processing speed scored over 11.5 points higher on the IDS-SR throughout follow-up than those with a low level of persistence (<25% of timepoints), and scored 4.5 points higher on the WSAS throughout follow-up than those with a low level of persistence (<25% of timepoints).Analysis of the individual THINC-itSensitivity analyses excluding the item of the IDS-SR which asks about concentration and decision making did not alter findings (see online Supplementary Table S1).Higher persistence of subjective cognitive difficulties measured via the PDQ-5 was associated with worse functional impairment across all domains of work and social adjustment (see online Supplementary Table S2). For objectively measured cognitive difficulties, only the highest persistence group showed significant associations across the domains of work and social adjustment.Increased persistence of attentional difficulty was particularly associated with worse work and private-leisure functioning. Those with the most persistent difficulty with working memory reported worse functioning in work, social-leisure, private-leisure, and relationships over time. Persistent problems with processing speed and executive function were associated with all functional outcomes: work, household, social leisure, private leisure and relationships.® modules, but small associations with the subjective PDS-5 measure. Of note, we identified substantially more people with highly persistent objectively-measured cognitive difficulties than self-reported with the PDQ-5, supporting previous suggestions that people may under-report their own cognitive function (Baune et al., Previous research has demonstrated discrepancies between subjective and objective measures of cognitive difficulty in people with a diagnosis of depression (Petersen, Porter, & Miskowiak, If we delve into these specific associations further, we see stronger patterns in certain modules. Those with the most persistent difficulties with working memory and executive function appear to rate their functional performance across most functioning domains as lower. Previous research has shown that concentration difficulties account for over 35% of impairment at work (Fried & Nesse, One of the strengths is our novel approach to data collection. We collected multiple cognitive assessments via smartphones, which overcome the time challenges of conventional clinic-based assessments (Matcham et al., ® tool provided less data for analysis out of all the measurements collected within RADAR-MDD (Matcham et al., The study does have some limitations. Although cognitive assessments may prove a useful predictor of depression and functional status outcomes the THINC-its.d. above/below normal population scores to determine the presence of cognitive difficulties across the cognitive modules, however this often resulted in extremely small group sizes. Our median scores across THINC-it® modules indicate worsened cognitive performance than recently reported in an analysis of healthy controls (Dalby et al., Another consideration is the nature of this study as a secondary analysis of an existing dataset, which was not powered to address this specific question. We used a well-defined threshold of scores ± 1® every 6-weeks, allowing for some standardisation of the duration between assessments across participants. However, our analysis cannot distinguish between the likely differences in performance between those who completed the assessment twice in 2 years, and those who completed it 10 times. Finally, although we have conceptualised the cognitive modules as separate, there is likely to be overlap between the cognition modules (Pan et al., The limitation of multiple cognitive assessments over long periods of time is the potential for practice effects: the tendency for individuals to perform better over time with repeated opportunities to practice the tasks (Wesnes & Pincock, Our work highlights several recommendations for future investigation. Replicating our findings in case control studies deliberately recruiting individuals with differing levels of cognitive difficulties could ensure comparisons are made across equal groups with sufficient statistical power. We hypothesise that withdrawal from functional activities, particularly social situations, due to difficulties with cognition, may reduce confidence in being able to cope with and get back into, those functional activities. Part of the solution may be increasing coping resources, e.g., through the flexible implementation of cognitive strategies through interventions such as cognitive remediation therapy (Wykes & Reeder, We have demonstrated that when asking people with depression directly about cognitive difficulty there is a relationship between persistent severity of depression and functional disability. We have shown that different elements of cognitive difficulty are differentially associated with worsened depression and function outcomes, with persistent challenges with working memory and executive function most consistently associated with poor outcomes. As we cannot untangle the direction of the relationships further research should explore interventions that target both cognitive and functional disability.


Understanding the mechanisms of microbiome assembly during host development is crucial for successful modulation of the gut microbiome to improve host health and growth. However, results from previous microbial intervention studies to improve swine growth have largely been inconsistent due to the constantly changing nature of the gut microbiota and limited longitudinal sampling. Detailed characterization of the swine gut microbiota through meta-analysis allows us to understand the dynamics of microbial community succession, as well as the transient and natural variations between time points and animals. A total of 3,313 fecal microbial communities from over 349 pigs covering 60 time points (from birth to market age) from 14 publications were included in this meta-analysis. Despite differences in animal breeds and management, generalizable patterns of community assembly were identified. Alpha diversity continuously increased during early stages of animal growth and more slowly in the later stages. Beta regression analysis revealed that more microbial taxa were enriched, while fewer were excluded by the gut microbiota. The microbial community structure also changed significantly between days at early ages and became more similar as pigs aged, as revealed by dissimilarity and distance metrics. Dirichlet multinomial mixtures analysis supported gradient microbial clustering in longitudinal pig fecal samples, and we found that the early samples spread to more clusters than those from older pigs. Random forest regression identified 30 operational taxonomic units as potential microbial biomarkers for modeling swine gut microbiota development. External validation showed this model could be generalized to future microbiome studies conducted in suckling and weaning pigs and adds to the toolkit to quantify community succession.The swine gut microbiome undergoes an age-dependent assembly pattern with a developmental phase at early ages and a stabilization phase at later ages. Shorter time intervals and a wider range of data sources provided a clearer understanding of the gut microbiota colonization and succession and their associations with pig growth and development. The rapidly changing microbiota of suckling and weaning pigs implies potential time targets for growth and health regulation through gut microbiota manipulation. Since swine gut microbiota development is predictable, swine microbiota age can be calculated and compared between animal treatment groups rather than relying only on static time-matched comparisons. The intestinal microbiota plays a vital role in both human and animal health. Mounting evidence has proven the associations between the human gut microbiota and immunity, metabolism, and health –4. DysThe longitudinal composition of the swine microbiota has most commonly been described at weekly or monthly intervals . Le ScieSimilarly, longitudinal studies have also determined the compositional and functional development of the human infant microbiota and have provided a foundation for mechanistic investigations of microbes and host health . HoweverThus far, studies of the community succession of the swine microbiota have only used large sampling time intervals, which could lead to inconsistencies between studies. High-resolution analysis with shorter time intervals is critical in determining the continual dynamics of microbiota composition rather than infrequent “snapshot” samples , 24. DifIn the current study, we performed an individual participant data (IPD) meta-analysis of fecal samples collected from commercial pigs in order to fill the knowledge gap that exists on the detailed succession of the swine gut microbiota. Overall, the swine gut microbial community develops in a consistent and predictable manner until a stable community is established. We also identified and utilized age-discriminant taxa to predict the microbiota maturation state and identified core microbial members that persist in the pig gut.Data from 14 studies published from 2019 to 2021 were included in this meta-analysis . There wP < 0.001; Generally, the age of the pigs was positively correlated with alpha diversity. Faith’s phylogenetic diversity index, which estimates the community phylogenetic diversity, increased over a large time span  explained by pig age, growth stage, study, hypervariable region sequenced, sequencing platform, and country of origin. Not surprisingly, the two most influential factors affecting the swine gut microbial structure were “study” and the growth stage of pigs  ordination plots in combination with permutational multivariable analysis of variance (PERMANOVA) and permutational multivariate analysis of dispersion (PERMDISP) on beta diversity distances between control samples from untreated pigs. We first assessed sources of heterogeneity using the variance  model, including both control and treated pigs from each study . The DMMTo circumvent the heterogeneity associated with including 14 independent studies, we also analyzed separately the three largest studies that had samples from all growth stages and found similar cluster distribution patterns  , 20, 37.After identifying clusters and the greatest contributing taxa within each cluster, we next sought to identify OTUs that could be used to discriminate pig age. We identified 30 OTUs correlated with pig age in the gut using random forest (RF) models .r = 0.99, R2 = 0.99) and test  data sets than the external validation , although there were significant (P < 0.001) linear correlations between the chronological age and he microbiota age observed in all three data sets. Based on the data distribution, we further split our external validation data set into early samples (age <80 days) and late samples (age >80 days), and the predictive performance was much improved in the early  compared to the late  samples . More robust predictive performance was also observed for early samples in training and test data sets, indicating that samples from early stages contributed more global variation in the entire data set (Table S10). Moreover, based on Pearson’s correlation coefficient and the accuracy of the range of predicted microbiota ages, models created using all time points had an advantage over models created with sparse time points or the single largest data set when applied to the external validation samples .The core microbiota refers to a group of consistent microbial taxa that are present across multiple hosts. The concept of a “core” microbiota in the swine gut is intriguing because it could lead to the identification of possible targets for dietary or pharmacological interventions in swine production systems. We chose to define the core microbiota as taxa present in at least 90% of all samples, an arbitrary but widely accepted threshold , 38, 39.Lactobacillus, Clostridium sensu stricto 1, Christensenellaceae R-7 group, (Eubacterium) coprostanoligenes group, Parabacteroides, Desulfovibrio, Blautia, Lachnospiraceae UCG-008, Lachnoclostridium, and Phascolarctobacterium were shared in the four stage-specific core groups . Next, we loosened the threshold to 50% to identify a lifelong core microbiota. The core genera that are present in at least 50% of the samples from all time points were Escherichia, Streptococcus, Lactobacillus, Clostridium sensu stricto 1, Bacteroides, Christensenellaceae R-7 group, Lachnoclostridium, and Faecalibacterium. Overall, our results extended previous definitions of the pig core microbiota  in the external validation data set, despite not being used to train the models. Those OTUs identified from the RF models either decreased or increased in relative abundance over time, indicating their marker function. Since intestinal microbiome development is a continuous process, future studies could benefit from comparison to a continuous reference (like our RF model) rather than snapshot comparisons to a treatment group. This approach has been adopted in human microbiome studies, and it has been reported to be important for human health –57. Fode novo analysis , i.e., re-analysis of the raw sequencing data  and the change of the sow’s milk composition over time . AnotherThe concept of enterotype has been highly discussed and debated in human microbiome clustering analysis , 67. ResFuture studies should focus on the development of the swine microbiome over time, rather than simply time-matched treatment comparisons. It would be intriguing to determine if the bacterial species represented by the age-discriminatory OTUs we identified from pigs  could contribute to pig recovery following biotic or abiotic challenges that cause developmental delays. We observed increasing trends in the relative abundance of most age-discriminatory OTUs, indicating limited-time persistence of early colonizers. The early colonizers may be due to milk selectivity and the later colonizers selected by the introduction of solid feed. The early colonizing microbiota showed immune activation function in humans and may exert a lifelong impact on host health , 70. It When assessing certain microbiota-derived interventions, it is important to focus on their dynamic influence rather than their cross-sectional impact. Previously, age was considered a key factor in swine gut microbiome development, but there was no clear consensus on how the microbial community develops over time in swine. The lack of universal developmental patterns limits our understanding of the swine gut microbiome and the application of microbiota-derived therapies or growth promoters. We filled this knowledge gap on the dynamics of the swine gut microbiota using continuous data by describing the microbial community with extensive data sets to determine that the swine gut microbiota undergoes predictable community succession, at least until 80 days of age, after which additional characterization of the microbiome is needed. The microbial community changes rapidly in the early stage followed by a slower succession as pigs age. The identified microbial biomarkers in each stage may play an important role in animal gut homeostasis and need to be further explored.https://www.webofscience.com/), PubMed (https://pubmed.ncbi.nlm.nih.gov/), and the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) databases in January 2022.Sequence data were obtained from 14 publications that included longitudinal sampling of the commercial pig fecal microbiota over at least three time points . To avoiMetadata are included in Table S1 and include country, pig breed, DNA extraction approach, PCR primers used, and sampling time. Only control samples from untreated pigs were used to determine the microbial community succession independent of treatment effects, unless otherwise stated. Time points with a sample size exceeding 50 were resampled to 50 samples to account for bias associated with the non-uniform distribution of samples between studies, unless otherwise stated .q-score function, with a minimum quality score threshold of 20  was used to remove the primers from these sequences . Then, rld of 20 .After dereplication with the dereplicate-sequence command, the quality-filtered reads were clustered into OTUs using the cluster-features-closed-reference function in the q2-vsearch plugin. Closed-reference clustering was performed at 97% identity against the SILVA (132 QIIME release) 97% OTU reference database . Reads tde novo chimera checking with the UCHIME-de novo command and excluded sequences containing chimeras using the q2-feature-table plugin with the filter-features command.A total of 94,467,293 quality-filtered sequences were obtained and clustered into 35,929 OTUs after closed-reference OTU picking. The closed-reference OTU picking workflow is a database-dependent strategy that clusters sequences into OTUs and assigns taxonomy to reference sequences using a pre-existing collection of reference sequences with known taxonomy. Lastly, we used Alpha and beta diversity analyses were conducted in QIIME2 using the q2-diversity plugin with core-metrics-phylogenetic command. In order to keep a reasonable number of samples for further analysis, samples were rarefied to 7,550 reads, which retained 90% of all samples, a sampling depth sufficient to observe differences between samples , especiaTo determine the key phases of microbiota progression, we used DMM clustering as an unsupervised method to bin samples based on the structure of the microbial community . The lowP/3, where P is the number of input OTUs). Data sets from two publications were randomly selected as external validation samples (229 samples), and the remaining samples from the other 12 publications were divided into a training set  and a test set (286 samples) based on the pig age using the “reateDataPartition” function in the “caret (version 6.0-90)” package. The “rfcv” function with 10-fold cross-validation was used to estimate the optimal number of top-ranking age-discriminatory OTUs required for prediction. To test the quality of the RF models created using varying amounts of training data, we also created RF models based on the single largest data set  . PERMANOs method . Beta res method . To eval


A new quinolone antibiotic called garenoxacin was developed in Japan. Garenoxacin is known to produce cutaneous adverse effects, particularly fixed drug eruption in Japan, despite several reports of cutaneous adverse events in English-language literature. However, English-language literature has not yet reported that fixed drug eruption is a common clinical manifestation of garenoxacin-induced drug eruption. In this article, we present a case of multiple fixed drug eruptions and review the literature on case reports of drug eruptions caused by garenoxacin. Garenoxacin is a new quinolone antibiotic developed in Japan . As a unA 25-year-old woman who had a sore throat was treated with acetaminophen, desloratadine, montelukast sodium, garenoxacin, and L-carbocysteine. A few hours later, she noticed erythema on the cheeks and lips Figure , as wellA physical examination revealed that a scaly pigmented round-form macule with black clasts was located on her right cheek Figure , which wA skin biopsy taken from the lip lesion revealed dyskeratotic keratinocytes in the epidermis and infiltrated lymphocytes into the epidermis and dermis Figure . Based oTo explore the causative agents, we first conducted lymphocyte stimulation tests because the multiple fixed drug eruption has been known as useful in identifying the causative agents; however, all the drugs at a concentration of 10% showed negative results. We then conducted patch testing and 10% of garenoxacin showed a positive reaction 48 hours after the patch testing Figure . The prePrednisolone 25 mg per day was administrated for three days with the discontinuation of garenoxacin. Mucosal erosions and skin erythema rapidly improved, and the prednisolone dose was decreased to 20 mg per day for seven days. One month after the start of treatment, steroids were tapered off. The patient was discharged from the hospital with a favorable clinical course without any recurrence of her skin eruption.Fixed drug eruption is recognized as repeatedly erythematous pigmented plaques following the same causative agents between 30 minutes and eight hours (on average two hours). Blisters occasionally occur on fixed drug eruptions  and can Garenoxacin is a representative causative drug causing fixed drug eruption. To clarify the characteristics of garenoxacin-related drug eruption, we reviewed all English and Japanese reported cases of drug eruption caused by garenoxacin Table  2,3,6-1-16-13. IGarenoxacin was first developed in Japan, and garenoxacin-related drug eruption has been mainly reported in Japan. The characteristics of garenoxacin-related drug eruption are specific and fixed drug eruption is a typical cutaneous adverse reaction. We reported a representative case and discussed the previously reported cases mainly described in the Japanese literature. Our literature review will provide useful information for clinicians to obtain a better understanding of fixed drug eruption as a representative garenoxacin-related drug eruption.


Deep brain stimulation (DBS) is an established and effective therapy for movement disorders. Here, we present a case of secondary myoclonus-dystonia syndrome following acute disseminated encephalomyelitis (ADEM) in childhood, which was alleviated by DBS. Using a patient-specific connectome analysis, we sought to characterise the fibres and circuits affected by stimulation.We report a case of a 20-year-old man with progressive dystonia, myoclonic jerks, and impaired concentration following childhood ADEM. Motor assessments utilising the Unified Myoclonus Rating Scale (UMRS) and the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) revealed a greater improvement in dystonia compared to myoclonus following adjustments of DBS parameters. These adjustments were based on visualisation of electrode position and volume of tissue activated (VTA) 3 years after surgery. A patient-specific connectome analysis using the VTA as a region of interest revealed fibre tracts connecting to the cerebello-thalamo-cortical network and the superior frontal gyrus in addition to basal ganglia circuits as particularly effective.Globus pallidus internus (GPi) DBS shows promise as a treatment for secondary myoclonus-dystonia syndromes. Personalised structural considerations, tailored to individual symptoms and clinical characteristics, can provide significant benefits. Patient-specific connectome analysis, specifically, offers insights into the structures involved and may enable a favourable treatment response. Whereas the exact pathomechanisms remain elusive, the thalamus and the basal ganglia are commonly affected . Involve2.We evaluated a 20-year-old male patient with a history of progressive dystonia, myoclonic jerks, speech difficulties, and impaired concentration. He was diagnosed with ADEM at the age of 10 in November 2009 and underwent several weeks of intensive medical therapy followed by months of rehabilitation. While initial symptoms  resolved, dystonic symptoms first appeared in December 2009, manifesting as involuntary movements in his right index finger while writing. Over the course of 3 years, the patient’s motor symptoms progressed beyond dystonia to myoclonic gestures.On initial examination, the patient presented with involuntary movements of the right arm and face, which were characterised by focal dysrhythmic jerks that were exacerbated by certain postures, movements, and stimuli. He also presented with multifocal dystonia of the right arm and foot. Brain MRI revealed lesions in the pons, cerebellum, thalamus, and periventricular white matter around the lateral ventricles .Despite the often self-limiting nature of ADEM, we hypothesised an association between focal demyelination in the thalamus  and braiThe patient was thus diagnosed with a secondary multifocal myoclonus-dystonia syndrome following ADEM. Dystonia and myoclonus had remained refractory to anticonvulsants  and anticholinergics (Trihexyphenidyl). Following thorough multidisciplinary evaluation, the patient decided to undergo unilateral lead placement in the left GPi using directional leads . DBS programming according to monopolar review revealed the largest therapeutic window when the current was steered in the anterior direction of the distal three-segmented ring contact. Using a top-down approach, we gradually increased the stimulation amplitudes to the maximum tolerated voltage while maintaining a frequency of 130 Hz and a pulse width of 60 μs. The patient was allowed to adapt DBS settings according to his preferences and was informed about the known long-term effects in dystonia. He deliberately chose programme 2  because it provided the most significant relief of motor symptoms .During 3 years of follow-up, the patient reported worsening of dystonia and myoclonic jerks. To address these issues, we introduced four new stimulation programmes. In view of reports indicating efficacy of higher stimulation frequencies and pulse widths in the treatment of dystonia , increasWe assessed motor symptoms employing the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) and the Unified Myoclonus Rating Scale (UMRS) with DBS turned off and on. This resulted in predominant improvement in dystonia  relative to myoclonus  after imaging-guided adjustments of DBS parameters. See 2.1.2 and 30 independent diffusion gradient directions. The DBS lead and VTA visualisation was carried out using the LEAD-DBS softwareMagnetic resonance imaging data were acquired 1 day before surgery on a 3 T scanner. The preoperative imaging protocol included b-values of 0 and 1,000 s/mmUtilising the DISTAL Minimal Atlas to define parcellation of grey matter structures , the VTA3.To the best of our knowledge, this is the first published case of secondary myoclonus-dystonia syndrome after ADEM successfully treated with unilateral GPi DBS. Adjustments of DBS parameters according to connectomic data for a modulation of distributed brain networks  has beenFor secondary movement disorders, growing evidence highlights a key role of alterations in multiple anatomical pathways . For dysWhile DBS targeting the posterior part of the ventrolateral thalamic nucleus (VLp) can effectively address myoclonus , 34, in 3.1.While this case report demonstrates the long-term effects of pallidal DBS in a secondary myoclonus-dystonia syndrome and thereby highlights the underlying networks, there are some limitations. Although imaging-guided optimisation of DBS parameters was carried out based on the VTA and its adjacent neuroanatomical structures, the stimulation parameters were not refined using the tractography results from the patient-specific connectome analysis. In addition, our preoperative imaging protocol prevented us from testing for abnormal kurtosis measures. Diffusion kurtosis imaging (DKI) has been shown to be more sensitive to microstructural changes in demyelinating disorders  by quantThe datasets presented in this article are not readily available because of ethical and privacy restrictions. Requests to access the datasets should be directed to the corresponding author.Ethical review and approval was not required for this case report in accordance with the local legislation and institution requirements. The patient provided written informed consent for the use of anonymized data for research purposes and for publication of any potentially identifiable images or data included in this article.AC and LB collected the clinical data and co-wrote the manuscript. AC performed the image analysis. MG, MB, and CN contributed to the data curation. LT and DP confirmed the diagnosis of the case and supervised the study. All authors contributed to the article and approved the submitted version.


Genetic biases in the human antibody repertoire result in publicly available antibody sequence datasets that contain many duplicate or highly similar sequences. Available datasets are further skewed by the predominance of studies focused on specific disease states, primarily cancer, autoimmunity, and a small number of infectious diseases that includes HIV, influenza, and SARS-CoV-2. These biases and redundancies are a barrier to rapid similarity searches and reduce the efficiency with which these datasets can be used to train statistical or machine-learning models. Identity-based clustering provides a solution; however, the extremely large size of available antibody sequence datasets makes such clustering operations computationally intensive and potentially out of reach for many scientists and researchers who would benefit from such data.Antibody Reference Clusters (AntiRef), which is modeled after UniRef, provides clustered datasets of filtered human antibody sequences. Due to the modular nature of recombined antibody genes, the clustering thresholds used by UniRef for general protein sequences are suboptimal for antibody clustering. Starting with an input dataset of ∼451M full-length, productive human antibody sequences, AntiRef provides reference datasets clustered at a range of antibody-optimized identity thresholds. AntiRef90 is one-third the size of the input dataset and less than half the size of the non-redundant AntiRef100.zenodo.org/record/7474336). All code used to generate AntiRef is available on GitHub (github.com/briney/antiref). The AntiRef versioning scheme (current version: v2022.12.14) refers to the date on which sequences were retrieved from OAS.AntiRef datasets are available on Zenodo ( The massive diversity of the human antibody (Ab) repertoire is produced initially by somatic recombination of germline gene segments. Considering both heavy and light chains, it is estimated that the recombination process can generate as many as 1018 unique Abs , enablinThese studies are hampered, by the scale of available Ab sequence data and the lack of standardized datasets of substantially reduced size that maintain an accurate representation of overall diversity. The availability of such datasets is vitally important; one particularly relevant recent example is the use of UniRef datasets  to trainThe Ab sequences used to construct AntiRef were downloaded from the Observed Antibody Space (OAS) repository  a complete VDJ region, (ii) no V-gene frameshifts, (iii) in-frame V and J genes, (iv) no stop codons, and (v) no ambiguous amino acids. After filtering, the dataset contained 260 373 862 heavy chains and 190 684 852 light chains, for a total of 451 058 708 sequences. Each sequence was given a unique identifier using Python’s built-in -c 0.8 and –cov-mode 0, which together require that linked sequences must align for at least 80% of the length of the longer sequence; and (ii) –min-seq-id <threshold>, which clusters sequences using the appropriate identity threshold for each AntiRef clustering operation. The sequence identity is computed across the entire aligned region, including gaps, meaning sequences that are otherwise identical but differ in length by N amino acids would be scored equivalently to two sequences of the same length with N mismatches. Following each iteration, representative sequences for each cluster were used as input for the subsequent round. This mirrors the strategy used by UniRef and ensures continuity of cluster names across all AntiRef datasets  over-represented. As a result, the general protein language model ESM-2 demonstrated marked improvement when trained with UniRef clusters rather than raw sequence data , providiTo determine the extent to which AntiRef clusters include different Variable (V) genes and/or V alleles, we sampled the 100 largest clusters from each AntiRef dataset and computed the fraction of each cluster encoding the single most common V allele in the cluster . As expeInspired by the usefulness of UniRef databases for various computational analyses of general proteins, AntiRef has been created to fill a similar role for Ab sequences. A series of AntiRef datasets have been generated using a nested clustering approach and Ab-specific identity thresholds. The uniqueness of antibodies within the general protein space necessitates the construction of Ab-specific resources like AntiRef. Proteins with high sequence similarity tend to have similar function, however, although similar Ab sequences are often functionally similar, a small number of differences in antigen recognition regions can result in highly divergent structural or functional properties. Additionally, the modular nature of Ab recombination means that tools and algorithms trained using general protein datasets may perform very well for the majority of the Ab protein encoded by these conserved, modular components but perform very poorly in the untemplated and often structurally labile antigen binding regions that determine Ab function.By making available all code needed to completely reproduce AntiRef, we hope to encourage future work extending AntiRef beyond human antibodies to additional species and T cell receptors.


Performance during risky decision making is one of the essential cognitive functions that is impaired in several psychiatric disorders including addiction. However, the cognitive mechanism and neural correlates underlying risky decision making in chronic pain patients are unclear. To our knowledge, this study is among the first to construct computational models to detect the underlying cognitive process of chronic pain patients during risky decision making.This study aimed at inspecting the significantly abnormal risky decision-making patterns of chronic pain patients and its neuro-cognitive correlates.In this case-control study, 19 chronic pain patients and 32 healthy controls (HCs) were included to measure the risky decision making in a balloon analogue risk task (BART). Optical neuroimaging using functional near-infrared spectroscopy, together with computational modeling, was carried out to systematically characterize the specific impairments based on BART.Computational modeling findings on behavioral performance demonstrated that the chronic pain patient group exhibited significant deficits in learning during BART (Long-term aberrant pain responses significantly disrupted the PFC function and behavioral performance in chronic pain patients. The joint behavioral modeling and neuroimaging techniques open a new avenue for fully understanding the cognitive impairment and brain dysfunction of risky decision making associated with chronic pain. In addition to the brain regions associated with pain pathways, regions of emotional response, including amygdala and nucleus accumbens, have also been observed to exhibit abnormalities in cases of chronic pain, in line with the findings from neuropsychiatric disorders.,Decision making constitutes an integral part of daily life. It involves a complex process whereby individuals try to fulfill their aims and expectations based on their judgments regarding rewards and risks under internal and contextual/social constraints.,In particular, the balloon analogue risk task (BART) has been widely used to measure risk-taking behaviors. BART is able to illustrate a real-world situation to identify an overall propensity for risk taking rather than a unique likelihood of engaging in a particular type of risky behavior.In addition, to investigate the neural correlates of risky decision making among chronic pain patients, functional near-infrared spectroscopy (fNIRS) neuroimaging was carried out. As a noninvasive optical neuroimaging technique, fNIRS measures the concentration changes of oxygenated hemoglobin (HbO) and deoxygenated hemoglobin (HbR) in brain tissue in response to neuronal activation. This technique has several distinctive advantages, including compatibility with other devices, low cost, robustness against motion artifacts, higher temporal resolution compared with functional magnetic resonance images, and higher spatial resolution compared with EEG.Therefore, this study aims to inspect the cognitive impairment associated with risky decision-making in patients with chronic musculoskeletal pain. It is hypothesized that risky decision making was significantly impaired in chronic musculoskeletal pain patients compared with that from the HCs. To test the hypothesis, concurrent behavioral modeling and fNIRS neuroimaging were performed to inspect the specific impairments and potential neural mechanisms during BART. In particular, using the computational model, nuanced cognitive processes during BART would be revealed, which could further relate to the abnormalities that contribute to the aberrant BART performance. Meanwhile, fNIRS recordings can capture the significant difference in brain activation between chronic pain patients and HCs. It is expected that this study might provide new insights into the cognitive neural mechanism associated with risky decision making in chronic musculoskeletal pain.22.1Nineteen chronic musculoskeletal pain patients . It took 8 to 12 min to finish the tests, and the reward was offered to each participant based on his or her performance in the task.2.3,As mentioned earlier, two computational models were chosen based on existing studies,2.3.1The re-parameterized four-parameter model (RFPM) was based on two crucial assumptions: (i) participants need to renew their belief about the balloon’s explosion probability after each trial and (ii) participants can determine the optimal number of pumps before each trial. According to previous reports,Based on the first assumption, As for the other assumption, participants have to determine the optimal pump number .2.4The Artinis device  was utilized to measure the HbO and HbR changes in PFC with a sampling rate of 50 Hz at two wavelengths of 760 and 850 nm. Specifically, two near-infrared light sources and eight detectors with an inter-optode distance of three cm yielded eight fNIRS channels. In addition, the Montreal Neurological Institute (MNI) coordinatesfNIRS data were preprocessed using NIRS-KIT,,To estimate the brain activation in the PFC during the risky decision-making task, a general linear model (GLM) at the individual level was utilized.2.5a priori power analysis was conducted.In accordance with a case-control study protocol, the current statistical analyses were done in R software 3.6 and MATLAB 2016. First, a two-way ANOVA revealed that the BART scores were significantly different  parameters were calculated to quantify describe the underlying cognitive process when the two groups carried out BART . It was 3.3To verify the corresponding neural response of BART, the brain activation regions in the PFC were identified by fNIRS neuroimaging. The pairwise e mapped  by adopt4,,,,,–Consistent with previous findings,,Importantly, through constructing the computational model, we found that EWMVM––Further, using fNIRS neuroimaging techniques, we identified decreased cortical activation in the PFC during risky decision making for the chronic pain patient group, compared with HCs. The PFC has been recognized to play a vital role in perceiving, modulating, and reappraising pain through various ascending and descending tracts.,The distinctive risky decision-making behaviors and deactivated PFC activity during risky decision making of chronic pain patients provide an effective index of automatically discerning the chronic pain patients from HCs and measuring the intervention efficacy. Several machine learning methods have achieved satisfactory performance in the diagnosis of chronic pain patients by combining cognitive tasks and neuroimage tools.In summary, through constructing computational models to quantitatively depict the underlying process of BART, we demonstrated that chronic pain patients have significant deficits in learning from the BART results and make decisions more randomly without deliberation. In addition, the deactivated brain activation in PFC was manifested for the chronic pain patient group, supporting the notion that long-term aberrant pain stimulus might damage the functions of the PFC. Combined evidence from behavioral and neuroimaging findings suggest that the altered risky decision-making abilities can provide new insights for discerning pain-related cognition impairment and understanding the cognitive neural mechanism of pain-related circuits, further achieving better intervention efficacy.