Background: Restrictive orders and temporary programmatic or ad hoc changes within healthcare and other supportive systems that were implemented in response to the COVID-19 epidemic in Malaysia may have created hindrances to accessing healthcare and/or receiving other supportive services for people who use drugs (PWUDs).Design: A primarily qualitative study has been conducted to evaluate how service providers and recipients were adapting and coping during the initial periods of the COVID-19 response.Settings: The study engaged several healthcare and non-governmental organizations (NGOs) in the peninsular states of Penang, Kelantan, Selangor, and Melaka.Participants: Medical personnel of methadone maintenance treatment (MMT) programs (n = 2) and HIV clinics (n = 3), staff of NGO services (n = 4), and MMT patients (n = 9) were interviewed using a semi-structured format.Results: Interviewed participants reported significant organizational, programmatic, and treatment protocols related changes implemented within the healthcare and support services in addition to nationally imposed Movement Control Orders (MCOs). Changes aimed to reduce patient flow and concentration at the on-site services locations, including less frequent in-person visits, increased use of telemedicine resources, and greater reliance on telecommunication methods to maintain contacts with patients and clients; changes in medication dispensing protocols, including increased take-home doses and relaxed rules for obtaining them, or delivery of medications to patients' homes or locations near their homes were reported by the majority of study participants. No significant rates of COVID-19 infections among PWUDs, including among those with HIV have been reported at the study sites.Conclusions: Although the reported changes presented new challenges for both services providers and recipients and resulted in some degree of initial disruption, generally, all participants reported successful implementation and high levels of compliance with the newly introduced restrictions, regulations, and protocols, resulting in relatively low rates of treatment disruption or discontinuation at the study sites. In response to the coronavirus COVID-19) threat, Malaysia imposed several phases of the Movement Control Order (MCO) nationwide, beginning on March 18, 2020 9 threat,. During Though these restrictive orders were intended to slow the spread of the COVID-19, and indeed they have shown considerable reduction of new infections in Malaysia, there is a concern that for people who use drugs (PWUDs), including those with substance use disorders (SUDs), the various types of measures to control or restrict peoples' movement, distancing or limiting social contacts, or restricting access to various social and healthcare facilities may have created particularly challenging hindrances to receiving social support or accessing healthcare and other supportive services –10.The present study aimed to collect information and qualitative and quantitative data on the potential impact of the MCO on PWUDs in Malaysia to evaluate how service providers and recipients of these services were adapting and coping during this period in Malaysia.The study combined a qualitative component, consisting of interviews with key personnel, service providers, and SUD patients receiving treatment in participating clinics, and a quantitative component based on data from pre-MCO and during MCO/CMCO/RMCO periods from the MMT program at Sungai Buloh Hospital (SBH) in Selangor.The study was reviewed and approved by the Human Ethics Committee of Universiti Sains Malaysia, Penang (protocol # USM/JEPeM/COVID19-30). A written informed consent was obtained from all study participants. No personally identifiable information has been collected from the interviewees and clinic records used in quantitative analyses were de-identified before accessing and analyzing.The study was conducted in several locations in the peninsular states of Penang, Kelantan, Selangor, and Melaka. Selection of study sites was determined by the availability of healthcare and/or non-governmental organizations (NGO) facilities that could be engaged in the study research protocol.Qualitative interviews collected information from healthcare and service providers, as well as patients with SUD receiving treatment or other services during the MCO/CMCO/RMCO periods from March 18 to August 31, 2020. Quantitative study component evaluated urine toxicology tests results collected before the MCO period (December 2019 to February 2020) and during the RMCO period (June 2020 to August 2020) at the MMT program at SBH in Selangor.One infectious disease MD physician and two MD general practitioners from the HIV clinic and the methadone maintenance treatment (MMT) clinic, respectively, in SBH, Selangor; one MD physician from MMT clinic in Masjid Tanah, Melaka; and one nurse from Hospital Raja Perempuan Zainab in Kota Bharu, Kelantan were interviewed. A total of nine MMT patients were interviewed: four from Masjid Tanah, Melaka, and five from Kota Bharu. One programme manager and one programme coordinator at the AIDS Action Research Group (AARG) NGO in Penang; one programme coordinator from the Insaf Murni NGO in Selangor and one programme coordinator from the SAHABAT NGO in Kelantan were also interviewed. Data collection methods.Qualitative interviews were based on a semi-structured interview guide developed for the current study. The interviews focused on the following domains of interest: (a) programmatic changes in healthcare policies and protocols implemented during MCO/CMCO/RMCO periods; (b) implemented operational changes at the point of care level and at supportive services facilities; (c) effects of the MCO/CMCO/RMCO restrictions and other implemented changes on provision of healthcare and supportive services; (d) effects of MCO/CMCO/RMCO restrictions and other implemented changes on patient access; and (e) effects of MCO/CMCO/RMCO restrictions and other implemented changes on substance use.One participant was interviewed over the phone, all other interviews were conducted face-to-face. The interviewers wrote down answers to all questions and took additional notes as needed. Study personnel adhered to COVID-19 related regulations implemented by the Malaysian government pertaining to body temperature checks and being interviewed about potential symptoms and health status upon entering healthcare facilities, wearing face masks and maintaining social distancing during the interviews.Deidentified urine toxicology test results for opiates, benzodiazepines, methamphetamine, amphetamine, and tetrahydrocannabinol collected routinely as part of clinical monitoring at the MMT clinic at SBH in Selangor between December 2019 and August 2020 were also obtained.The analyses focussed on: (a) identifying information on changes in policies, protocols, operating procedures, and implemented practices, and (b) on evaluating potential impact of these changes and of COVID-19 related restrictions on healthcare access, and substance use among PWUDs during the MCO, CMCO, and RMCO in Malaysia.Collated notes from all qualitative interviews were reviewed by the study research group to identify informational content  and analyzed thematically to identify common patterns pertaining to impact, adaptation, and coping both on organizational and individual levels.Descriptive analyses were conducted using MMT clinic records data. The overall rates of urine toxicology test results positive for any illicit substances during each month of pre-MCO (December 2019 to February 2020) and RMCO (June 2020 to August 2020) were calculated, tabulated, and compared.The interviewed physicians reported that between January 2020 and August 2020 there were 131 and 78 active patients, respectively, in Masjid Tanah, Melaka and SBH, Selangor MMT clinics. In both clinics, there were no reported COVID-19 infections among their MMT patients.Prior to the MCO, take-home doses of methadone were provided according to the national guidelines to patients who were considered to be in a stable recovery, as determined by negative urine toxicology tests conducted randomly, at least once a month. Patients with continuous urine tests negative for all tested illicit substances  for at least 3 months were eligible for take- home methadone doses. The national guidelines for prescribing take home doses, allowed for eligible patients to initially receive 3 to 4 days of take-home doses, and subsequently the number of doses could have been increased for up to 2 weeks maximum, for patients who continued with a stable recovery . In bothDuring the MCO, urine testing was suspended until June 2020 at the SBH MMT clinic, but not at Masjid Tanah clinic in Melaka. Methadone take-home dose regulations were relaxed in both clinics participating in this study. Almost all patients in both clinics received take-home doses. Those who previously did not receive take-home doses started receiving a 1-week supply of methadone daily doses, and those previously on weekly take-home regimen, were receiving a 2-week supply of take-home methadone doses. Take home doses for both clinics were dispensed in individual bottles for each day of dosing. Patients were instructed to consume one bottle each day and return the empty bottles when coming to the clinic for their next take-home doses supply. The clinic staff has not collected any self-report on medication adherence, due to the brevity of clinic visit during the MCO.Only patients who were newly admitted to the MMT program during the MCO were required to come to the clinics daily during their initial dose titration period. However, there were very few new patients enrolling during the MCO. Only one new patient was reported in the Masjid Tanah, Melaka MMT clinic. No new patients were admitted during the MCO, CMCO, and RMCO at the MMT clinic at SBH.Interviewed MMT personnel indicated that they would prefer to continue with the relaxed rules for the methadone take-home dosing to continue even after the COVID-19 restrictions are ultimately lifted. As of November 2020, The Masjid Tanah MMT clinic in Melaka continues to provide take-home methadone doses to the majority of their patients. The MMT clinic at SBH returned to the pre-MCO regulations regarding take-home dosing in July 2020.Interviewed personnel reported that some patients missed their clinic visits and medication pick up visits during the MCO, CMCO, and RMCO, but beginning in July most of these patients reengaged with their clinics.Before the MCO has been declared, the SBH in Selangor had the largest HIV treatment programme in Malaysia, with a census of over 9,000 HIV patients. Coinciding with the declaration of the MCO the hospital has been designated as the primary treatment and coordinating center for COVID-19 patients. It has been reported that it had treated approximately 5,000 COVID-19 cases and there was a total of 12 COVID-19 related fatalities reported by the time of the study qualitative interviews. Among all HIV patients at the SBH, only one was reported to become infected with the COVID-19, received the same course of treatment as other COVID-19 patients, and subsequently fully recovered without any COVID-19 related sequalae.There were significant programmatic, structural/facilities, and organizational changes implemented to decrease concentration/congestion of people on the hospital grounds and to follow newly implemented social distancing rules, as well as to accommodate the new role for the hospital and to facilitate care for the expected influx of COVID-19 patients. The HIV in-patient ward was converted into an inpatient COVID-19 treatment ward. Other wards were also converted or designated to treatment of COVID-19 patients as needed. Existing HIV in-patients were transferred to other wards within the hospital, with some patients transferred to different hospitals or facilities, while some of the HIV outpatients who were assessed to require more vigilant care were admitted as inpatients. The SBH stopped accepting new non-COVID-19 with the exception of any urgent walk-ins. All new cases were referred to other hospitals. The SBH began accepting new non-COVID-19 patients around early to mid-June, after interstate travel was permitted. Patients traveling from other states received letters to certify their travel for important health related reasons. Overall, the interviewed healthcare professionals stated that the greatest challenge in maintaining clinic services was fatigue and COVID-19 case overload due to staffing shortages.EZ Clinic, aimed to ease patient flow through the on-site HIV clinic and to remove some of the challenges of healthcare access, by reducing delays in patient-provider contacts and reducing travel and time burden of an in-person visits for patients who could utilize the telemedicine service. Through the EZ Clinic healthcare providers were able to conduct a rudimentary patients evaluation, review laboratory test results, and provide a consultation for their patients.During the MCO, HIV patients who were determined to be clinically stable had their previously scheduled on-site face-to-face medical evaluation appointments with the clinic personnel canceled or postponed by 1 month. Additionally, a telemedicine consultation service offered to clinically stable HIV patients receiving ART operating at the SBH since 2017 continued during the MCO, CMCO, and RMCO periods. This service, called EZ Clinic were more likely to maintain regular contact with their treatment providers. On the other hand, patients who were not utilizing the EZ Clinic were more likely to miss their evaluation appointments during the MCO, CMCO, and RMCO periods.It was reported that patients registered with the For patients scheduled for an in-person visit, the HIV clinic nurse called the patients ahead of their appointment to evaluate their current health status before deciding if they need to come to the clinic. If the patients were clinically stable and generally doing well, they were asked not to come for their scheduled appointment. Patients attending their scheduled appointments in-person were not allowed to be accompanied by family members, which was very common before the MCO. All laboratory tests for stable patients were suspended during the MCO.Prior to MCO, patients who were receiving Antiretroviral Therapy (ART) were required to come to the on-site pharmacy to receive a monthly supply of ART medications. During the MCO, ART patients were given three options: sending/mailing their medication to their home or to the healthcare facility that was nearest to their residence; or drive through pharmacy pickup service at the hospital; or a walk-in pick up of prepared medication supply at the hospital lobby. No medication shortages were reported during the MCO, CMCO, and RMCO periods.Insaf Murni, an NGO that provides HIV-related services to key populations, operates in two towns within the Selangor state: Klang and Kajang. The AIDS Action Research Group (AARG) operates in Penang, and provides a broad range of services, including needle and syringe services and HIV testing and counseling at sites on the island and at the mainland. SAHABAT NGO operates in Kota Bharu, Kelantan and offers needle and syringe services, HIV testing and counseling and operates a home shelter for PWUDs.The interviewed NGO staff reported that a day before MCO was implemented, outreach workers from Insaf Murni have distributed a three-week supply of needles and syringes at their community distribution locations frequented by the PWUDs. During the MCO, CMCO, and RMCO, their organizations temporarily stopped providing counseling, community HIV testing, and drop-in services to the clients. All NGOs reported that they increased their needle and syringe package for clients from 1 week to 2 or 3 weeks supply and added face masks and disinfectants/sanitisers to the packages. Clients came to the organization dispensing sites in the community to pick up their packages.During the MCO period, new clients who were referred by existing clients were registered through phone calls, rather than through in-person visits. An increase in the number of PWUDs interested in being referred to MMT during the MCO has been reported. Insaf Murni NGO also reported an increase in request for HIV and Hepatitis testing among men who have sex with men (MSM) and from the transgender community during the MCO, CMCO, and RMCO periods.During the MCO, the government begun offering financial assistance to people who lost their jobs and a 6-month property and vehicles loan moratorium was introduced. The NGOs started to help their clients to complete the necessary application documents and assisted them in the application process. One NGO have also reported to provide food to 70 transgender people by delivering the food packages to their homes and to 120 PWUDs by delivering the food to the health clinic.Starting in May 2020, during CMCO, outreach workers at Insaf Murni restarted to transport clients to a health clinic for Hepatitis C treatment. Their outreach workers were provided with the sets of personal protective equipment (PPE) including a face mask, eye protection, isolation gown, and gloves for their off-site travel and community work. Outreach workers have used this opportunity to restart distribution of needles and syringes at locations frequented by PWUDs, within the 15 km radius of the two towns where this NGO has been operating. During RMCO, outreach workers continued engaging with PWUDs including referrals to MMT treatment and provision of 3-week supply of clean needles and syringes. Counseling sessions and HIV testing for PWUDs resumed in August 2020.Collection of used needles and syringes, community HIV testing and counseling programs, and narcotic-anonymous meetings were suspended. Collection of used needles and syringes resumed at Insaf Murni during RMCO, but the rates of returned needles and syringes dropped from pre-MCO 75% to ~30%.Some of the commonly reported challenges faced by the NGOs during the MCO, CMCO, and RMCO were difficulties reaching out to their clients, especially those who were living further away from the NGOs operating sites. To reach the clients living outside 10–15 km radius from the sites, the outreach workers needed to obtain a permission from the police. NGOs' case workers were also restricted in accompanying clients for their healthcare appointments. Some of the interviewed NGOs' staff remarked that during the initial stages of the MCO they were worried about potential shortages of needles, syringes, and other supplies due to the overall disruption in the supply chains in the country. However, no major shortages of such supplies were reported during the interviews.Five of the nine interviewed MMT patients were also receiving ART, and three of these were additionally receiving Hepatitis C treatment during the time of their study participation. All five MMT patients on ART were residing in the SAHABAT NGO shelter home in Kota Bharu, Kelantan. Prior to MCO, residents of the shelter home received weekly counseling. However, this service was suspended when the MCO was declared, as there were local travel restrictions preventing counselors from traveling to the shelter. The on-site staff of the shelter home continued to help the patients to ensure daily ART and Hepatitis C medication adherence and took them for scheduled follow up visits with their HIV clinic treatment providers as their HIV clinic in Kelantan continued to provide in-person services for patients residing in the shelter home.The interviewed patients confirmed that after the MCO has been implemented take-home doses were given to patients previously on a daily dosing regimen and those already receiving take-home methadone doses became eligible for up-to 2 weeks of methadone take-home dosing. One patient expressed a concern regarding his take-home doses. He said that he did not have a proper place to store the medication as he lived with two younger siblings and nephews and nieces. He was worried that they may accidentally consume his medication even though he kept it in a locked drawer.Interviewed patients reported that their respective MMT clinics implemented body temperature checks while entering the clinics, and social distancing rules on the clinics' grounds. They were also required to register in the national COVID-19 contact tracing application. Overall, all patients reported that generally they have not had significant problems in getting their supply of medications and their treatment was not interrupted throughout the MCO period.The interviewed patients expressed mixed views about availability of street drugs during the MCO. Some stated that the price of a packet of street heroin was unchanged while the quantity in each packet was somewhat reduced. Others, stated that the price of heroin increased during the MCO. They also expressed different opinions regarding availability of street drugs: some said that the supply/availability was reduced, while others reported no perceived changes in the supply or availability.This primarily qualitative study evaluated whether the MCOs imposed in Malaysia in March of 2020 in response to the COVID-19 epidemic and the related changes in healthcare and social support services created particularly challenging hindrances for PWUDs. The study collected semi-structured interviews with medical personnel of healthcare services, staff of NGOs, and MMT patients in the peninsular states of Penang, Kelantan, Selangor, and Melaka.While PWUDs, especially those who use opioids and amphetamine-type-stimulants (ATS), are vulnerable to respiratory and pulmonary health problems and they were feared to be at increased risk of infection and high rates of treatment discontinuation during the COVID-19 pandemic , no signInterviewed participants reported significant organizational, programmatic, and treatment protocols related changes implemented within the healthcare and support services in addition to nationally imposed MCOs. The main changes aimed to reduce patient flow and concentration at the on-site services locations, including postponing, or less frequent scheduling of in-person visits, especially for patients determined to be clinically stable. A greater utilization of telemedicine resources and greater reliance on telecommunication methods instead of in-person visits or contacts to maintain therapeutic or service engagements with patients and clients was also commonly reported. Both MMT programs and HIV clinics implemented significant changes in medication dispensing protocols, including relaxed rules for patients to obtain take-home doses, increases in the duration of take-home doses, and delivery of ART medications to patients' homes or locations near their homes. While these changes were meant to be temporary, in some study locations the modified/relaxed medication protocols were still in place after the study completion and may continue to be utilized in the future. In particular, despite the relaxation of the rules for eligibility of methadone take-home dosing, neither healthcare professionals nor patients reported significant challenges resulting from the expansion of methadone take-home regimens at the participating MMT clinics. Urine toxicology data obtained from the MMT clinic at SBH see  indicateInterviewed staff of NGOs reported challenges in accessing some clients, especially in locations further away from their organizations operation sites, primarily due to travel restrictions. They also reported temporary discontinuation of some of their services, including HIV testing and counseling, and any services necessitating face-to-face or close interaction with the clients. Other services, including needle and syringe distribution continued without major disruptions, due to procedural changes, adjustments, and adaptations. All needle and syringe programs reported providing increased number of needles and syringes in their distribution packets, and providing additional COVID-19 related supplies, such as face masks and disinfectants. Some NGOs also reported initiating additional services that were not typically offered during the pre-MCO period, for example, assistance with applications for new government assistance programs, or food distribution.Overall, no major or only transient disruptions in provided healthcare and other supportive services were reported by the interviewed healthcare providers, NGOs' staff, as well as MMT patients. Based on the conducted interviews and evaluation of available clinic records, the present study has not obtained any evidence of substantially increased rates of treatment or service discontinuation. Some increases in services demands  were also reported. Interviewed participants reported challenges related to travel/movement restrictions, and concerns about potential adverse effects of the disruptions in the supply chains on availability of medications and service supplies, however the study participants have not reported medication shortages or other significant treatment or supporting services disruption.Due to COVID-19 response burden on healthcare and other social services, as well as travel restrictions being still in place when the study was conducted, the study was able to engage only a limited number of services, and a relatively small number of healthcare providers, NGO staff, and patients were enrolled. Study findings are based primarily on qualitative interviews with only limited quantitative data obtained and analyzed. Consequently, the study findings represent only a snapshot picture. A broader range of changes and adaptations were likely being implemented in different locations throughout Malaysia in addition to the nationwide imposed MCOs.Despite these limitations, the study provides an overview of successful changes and adaptations that were implemented in Malaysia in response to the COVID-19 pandemic and outlines their potential impact on provision and access to healthcare and other supportive services for PWUDs in Malaysia. The study findings may inform future responses to potential crises and hindrances concerning provision of healthcare and social support services for PWUDs in Malaysia and other countries in the region.The reported changes and adaptations introduced to cope with the COVID-19 pandemic in Malaysia presented new challenges for both service providers and recipients and resulted in some degree of initial disruption. However, generally, all participants reported successful implementation of the changed or newly implemented procedures or protocols and high levels of compliance with the newly introduced restrictions, regulations, and protocols. The reports collected during the study indicate that both the personnel and patients or clients receiving services at the evaluated services were able to adapt well to the changes, resulting in relatively low rates of treatment or service disruption or discontinuation at the study sites.The data that support the findings of this study are available on request from the corresponding author.The studies involving human participants were reviewed and approved by Human Ethics Committee of Universiti Sains Malaysia. The patients/participants provided their written informed consent to participate in this study.NAMS, W-TC, NMZ, and DS were involved in the data collection. BV and MCC conducted the data analysis and drafted the manuscript. All authors contributed to the design, conceptualization of the study, revised and edited the manuscript, contributed to the article, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Growth factors play a vital role in cell proliferation, migration, differentiation and angiogenesis. Autologous platelet concentrates which contain high levels of growth factors are used in many fields of dentistry. The current review is designed to provide clinical information regarding the use of three autologous platelet concentrates techniques in periodontal regeneration and facial rejuvenation: platelet-rich plasma, platelet-rich fibrin and concentrated growth factor techniques. The aim is to provide the clinician with an up-to-date overview of autologous platelet concentrates evolution over the past decade, clinical indications for use and advantages and limitations of each technique. This article was written in clinical orientation and is designed to provide clinicians with reliable and useful information applicable to their clinical work. Overall, platelet-rich plasma is mainly used in cases of hard and soft tissue procedures, while platelet-rich fibrin is used in gingival recession and treatment of furcation and intrabony defects; concentrated growth factor is mainly used in bone regeneration. In the field of facial rejuvenation, the use of platelet-rich plasma promotes tissue remodeling in aged skin and may be used as an adjuvant treatment to lasers; platelet-rich fibrin holds significant potential for stimulated dermal augmentation, and concentrated growth factor treatment could improve the survival and quality of fat grafts.Growth factors (GFs) play a vital role in cell proliferation, migration, differentiation and angiogenesis. Autologous platelet concentrates (APCs) which contain high levels of GFs make them especially suitable for periodontal regeneration and facial rejuvenation. The main generations of APCs presented are platelet-rich plasma (PRP), platelet-rich fibrin (PRF) and concentrated growth factor (CGF) techniques. The purpose of this review is to provide the clinician with an overview of APCs’ evolution over the past decade in order to give reliable and useful information to be used in clinical work. This review summarizes the most interesting and novel articles published between 1997 and 2020. Electronic and manual searches were conducted in the following databases: Pubmed, Scopus, Cochrane Library and Embase. The following keywords were used: growth factors, VEGF, TGF-b1, PRP, PRF, CGF and periodontal regeneration and/or facial rejuvenation. A total of 73 articles were finally included. The review then addresses the uses of the three different techniques in the two disciplines, as well as the advantages and limitations of each technique. Overall, PRP is mainly used in cases of hard and soft tissue procedures, while PRF is used in gingival recession and the treatment of furcation and intrabony defects; CGF is mainly used in bone regeneration. The use of platelets for regenerative medicine has increased in recent years. Platelets, which contain growth factors, play major roles in cell migration, proliferation, differentiation and angiogenesis and are associated with the tissue regeneration process. Autologous platelet concentrates (APCs) are produced by the centrifugation of venous blood at different speeds and the use or non-use of thrombin and anticoagulant. As a result of these processing protocols, a fibrin clot is formed that contains platelets and leukocytes . The maiThere is also a mechanical impact of biomaterials on cell regeneration, repair and homeostasis. As previously described by Kraft et al., dental pulp stem cells (DPSCs) have the potential to repair and generate teeth and bone . DPSCs cThe term “dentinogenesis” refers to the complex dentin pulp which responds to external mechanical stress and repairs itself by forming dentin . The pulFurthermore, stem cells in the oral cavity express mesenchymal cell-like features and are attracting interest due to the fact that they can be easily isolated from teeth extracted due to irreversible periodontitis or for orthodontic reasons and also because of the large amount of cells that can be produced ,10. DiffThe current review will discuss the main APCs: PRP, PRF and CGF. PRP, which was first described by Whitmen et al., is prepared by the centrifugation of autologous whole blood together with thrombin and calcium chloride, to form a “platelet gel” . The secIn 1997, Whitman et al. published the first article on the use of platelet-rich plasma (PRP), the first generation of APCs, in oral and maxillofacial surgery. During the preparation of PRP, xenogeneic thrombin and anticoagulant were added. This technique uses exogenous materials and might cause an immunologic and infectious response, making its use controversial ,18.PRP plays a vital role in wound healing. The wound-healing process can be divided into three stages: biochemical activation, cellular activation and cellular response. First, there is a conversion of the mechanical injury into biochemical signals. This cascade is triggered by the Hageman factor in the serum. As a result of the disruption of microcirculation, the plasma comes into contact with tissue proteins and the basement membrane, activating the Hageman factor and platelets. The clotting cascade enables fibrin to facilitate homeostasis, and it activates thrombin. Thrombin, calcium chloride and ADP trigger the activation of platelets, leading to the release of alpha granules from platelets, with the subsequent secretion of a large variety of growth and differentiation factors .The complement cascade also includes the release of substances that are important for wound repair. During this process, bradykinin is produced, which causes vasodilatation and the activation of plasminogen to produce plasmin, which degrades the fibrin. The fibrin degradation causes monocyte migration and vasodilatation. The third stage is the cellular response. In this stage, GFs are released from platelets. These GFs signal the local epithelial and mesenchymal cells to migrate, divide and enhance the synthesis of the collagen matrix. The platelet count in PRP is 338% of the platelet count of the whole blood . PRP enhIn the pre-operative period, 450 mL blood is collected in a sterile centrifuge tube, containing citrate–phosphate–dextrose solution (as anticoagulant). First, it is centrifugated  at 5600 rpm. The result of this stage is the separation into two layers: first layer—platelet-poor plasma (PPP); second layer—red blood cells (RBCs) and buffy coat, which contains platelets and white blood cells (WBCs) see . Only thIn 2000, Choukroun et al. reported on a new alternative to PRP: platelet-rich fibrin (PRF) ,23. ThisAs described by Chouckroun et al., IV blood is collected in 10 mL tubes with no anti-coagulant addition; it is then centrifugated at 3000 rpm for 10 min. At the end of the procedure, three layers are obtained: 1. Bottom—RBC layer; 2. Middle—Fibrin clot layer (PRF); 3. Top—serum layer (PPP) see . As mentMiron et al. published a modification to PRF: a liquid formulation of PRF injectable PRF (i-PRF) with no use of anticoagulants. As compared to PRP, after 10 days, i-PRF released higher levels of GFs such as IGF-1, EGF, PDGF-AA/AB. Furthermore, i-PRF induced the highest fibroblast migration, while PRP induced higher levels of cell proliferation . FujiokaIn 2006, Sacco  reportedAs described by Bozkurt et al., IV blood is collected in two 10 mL glass-coated plastic tubes with no anticoagulant addition. The tubes are immediately centrifuged  in the following manner: 30’’ acceleration, 2’ 2700 rpm, 4’ 2400 rpm, 4’ 2700 rpm, 3’ 3000 rpm and 36’’ deceleration until end. At the end of the procedure, four layers are obtained from bottom to top: RBC layer, GF and stem cell layer (CGF), Buffy coat layer, serum layer (PPP) see . Then, tThe cementum, gingiva, periodontal ligament (PDL) and alveolar bone serve as the tooth-supporting tissues (periodontium). Periodontitis is a chronic multifactorial inflammatory disease which is primarily characterized by the destruction of alveolar bone and tooth-supporting connective tissue, which is manifested by a loss of clinical attachment, presence of periodontal pocketing and bleeding on probing . Lack ofIn recent years, there has been an increase in the use of the techniques in the field of skin rejuvenation due to the high concentration of growth factors in platelets. APCs are used in facial rejuvenation of the periorbital area and wrinkles, the treatment of acne scars and in lip augmentation. Kim et al. demonstrated that PRP stimulates dermal fibroblast proliferation, collagen synthesis and matrix metalloproteinase expression, thereby aiding facial rejuvenation . There aThis narrative review is designed to provide the clinician with an overview of APC evolution over the past decade. The uses of the different methods will be elaborated, to provide the clinician with reliable and useful information for clinical implementation and to consider novel uses of the three techniques in the fields of periodontal regeneration and facial rejuvenation. To the best of our knowledge, this is the first time in the literature that a narrative review article is devoted to discussing the uses of the three APC methods in the areas of periodontal regeneration and skin rejuvenation, summarizing the indications for using each method, as well as advantages and limitations, and presenting the reader with the most up-to-date information, mostly collected over the last decade.The Pubmed, Cochrane Library, Scopus and Embase databases were searched from January 1997 to December 2020 to find published studies on the effects of different autologous platelet concentrates on periodontal regeneration and facial rejuvenation. see  The keywThe following criteria were applied for APCs in periodontal regeneration: Study design: randomized controlled trials (RCTs), cohort studies, cross-sectional studies only in the English language;Population: only studies on humans, with a minimum sample size of 10 patients and no restriction in terms of patient ages;Intervention: regenerative periodontal surgery on interproximal bony defects (IBD) and furcation defects (FD);Types of outcome: probing pocket depth recovery (PPDR) and clinical attachment level gain (CALG).Inclusion criteria:Absence of baseline data before periodontal surgery;Patients with systemic diseases or craniofacial anomalies;No training in oral hygiene;Follow-up < 6 months.Exclusion criteria: The following criteria were applied for APCs in facial rejuvenation: Study design: randomized controlled trials (RCTs), cohort studies, cross-sectional studies, case reports and case series only in the English language;Population: only studies on humans; for CGF, studies on animals were included;Intervention: facial skin rejuvenation, facial wrinkles, atrophic acne scars;Types of outcome: clinical or histologic evaluation.Inclusion criteria:Hair and nail restoration;Patients with alopecia;Review articles.Exclusion criteria: For the manual search, we selected six journals . In addition, the references of the selected articles were evaluated to find additional publications by manual searches.The aim of the present study was to evaluate the state of the art in the use of autologous platelet concentrates in periodontal regeneration and facial rejuvenation. The electronic search identified 741 references see . CitatioSeven clinical studies on PRP use for infrabony defects were included in the current review see . Some stSince 2009, the use of the PRP technique has diminished, as demonstrated in Pradeep et al.  and BajaRegarding CGF, there are limited studies available with relation to periodontal regeneration. Only one study reported the effect of CGF in infrabony defects. The authors concluded that CGF can enhance bone regeneration and reduce the depth of periodontal intrabony defects. When combined with a xenograft, CGF might be a superior scaffolding material . CGF preAs mentioned in The current narrative review includes RCT studies, clinical studies, cross-sectional studies, case reports and case series. The aim was to provide clinicians with up-to-date information about the use of PRP, PRF and CGF in dentistry, including the clinical indications, advantages and limitations of each technique. This article has a clinical orientation and was written in a manner designed to provide clinicians with reliable and useful information applicable to their work and interest in novel uses of the three techniques in the field of periodontal regeneration and facial rejuvenation.Periodontal regeneration, following destructive episodes of periodontal disease, must involve not only the affected alveolar bone but also the periodontal ligament and the root cementum .The three techniques described in this article differ in GF amounts . Mazuki As previously described in Since 2009, the use of the PRP technique has diminished, as demonstrated in Regarding CGF, there are limited studies available in relation to periodontal regeneration. Its use has been proposed in dentistry for various situations, ranging from the filling of extraction sockets  to the fIn recent years, there has been increased use of the techniques presented in the field of facial rejuvenation. The added value of using these techniques is the ability to deliver a high amount of GF to the scar, acne, periorbital fine line and pigmentation target areas. The alternatives for facial rejuvenation are hyaluronic acid injections and fat grafting. The former is expensive and its effects last only up to 12 months, and the latter can cause unpredictable swelling. The periorbital region is an area of the face that enables us to estimate a person’s age and has aesthetic and beauty aspects. This area is prone to pigmentation, wrinkles, erythema, xerosis, decreased skin elasticity and melanosis, all of which corelate to the person’s age. PRP enhances rejuvenation by inducing collagen synthesis and dermal fibroblast proliferation. Facial aging is caused by extracellular matrix (ECM) alterations and poor fibroblast proliferation.Samadi et al. published a review regarding the multiple clinical uses of PRP for aesthetic and regenerative medicine . Mayes eThere are facial rejuvenation procedures that combine PRP and fat grafting to achieve facial volume. Rophael et al. indicated that the angiogenesis benefit from PRP may explain the mechanism underlying fat graft retention, especially during the ischemia period after fat injection . Cameli This narrative review is designed to provide clinical information regarding the use of three APC techniques in periodontal regeneration and facial rejuvenation: PRP, PRF and CGF. The advantage of the APCs is the fact that a large amount of GF can be delivered to the target site and encourage angiogenesis and wound healing. PRP is the most established among the techniques reviewed in this article and is used in soft and hard tissue repair. At early time points, PRP provides more rapid delivery of GFs to the target site as compared to PRF and CGF. As a result of the addition of PRP to a bone autograft, dense and mature bone is formed. However, the transmission of infectious diseases and coagulopathies serves as a crucial limitation to the PRP technique and should be taken into consideration. In contrast to PRP, PRF and CGF require only centrifuged autologous blood and therefore provide immunological biocompatibility. PRF is effective mostly for soft tissue repair such as gingival recession coverage and furcation defects and is also used for intrabony defects. CGF is used in oral surgery, mostly for hard tissue regeneration. This narrative review also discussed the use of the three APC techniques in facial rejuvenation. The added value of using these techniques is the ability to deliver a high number of GFs to the target site. The use of PRP promotes tissue remodeling in aged skin and may be used as an adjuvant treatment to lasers. PRF holds significant potential for stimulated dermal augmentation. CGF treatment could improve the survival and quality of fat grafts. As this is a narrative review, this serves as a limitation of this study. Thus, interpretation should be made with caution. Future high-quality studies, e.g., randomized controlled trials, should be conducted; future systematic reviews and meta-analyses regarding the current review topic are also warranted.


Nonsense-mediated mRNA decay (NMD) is a highly conserved cellular surveillance mechanism, commonly studied for its role in mRNA quality control because of its capacity of degrading mutated mRNAs that would produce truncated proteins. However, recent studies have proven that NMD hides more complex tasks involved in a plethora of cellular activities. Indeed, it can control the stability of mutated as well as non-mutated transcripts, tuning transcriptome regulation. NMD not only displays a pivotal role in cell physiology but also in a number of genetic diseases. In cancer, the activity of this pathway is extremely complex and it is endowed with both pro-tumor and tumor suppressor functions, likely depending on the genetic context and tumor microenvironment. NMD inhibition has been tested in pre-clinical studies showing favored production of neoantigens by cancer cells, which can stimulate the triggering of an anti-tumor immune response. At the same time, NMD inhibition could result in a pro-tumor effect, increasing cancer cell adaptation to stress. Since several NMD inhibitors are already available in the clinic to treat genetic diseases, these compounds could be redirected to treat cancer patients, pending the comprehension of these variegated NMD regulation mechanisms. Ideally, an effective strategy should exploit the anti-tumor advantages of NMD inhibition and simultaneously preserve its intrinsic tumor suppressor functions. The targeting of NMD could provide a new therapeutic opportunity, increasing the immunogenicity of tumors and potentially boosting the efficacy of the immunotherapy agents now available for cancer treatment. Nonsense-mediated mRNA decay (NMD) pathway is a protection mechanism of eukaryotic cells, being responsible for the identification and degradation of mRNAs with aberrant mutations. In this way, NMD prevents the production of altered polypeptides with potentially toxic dominant-negative activity . AlthougThe detection of a transcript bearing a premature termination codon (PTC) is the best characterized NMD function. However, the NMD machinery is not only able to target mutated disease-causing mRNAs, but also to physiologically regulate about 10% of the transcriptome of human cells . The NMDXenopus model [The EJC-dependent NMD has been considered the only NMD activation mechanism for a long time. According to this mechanism, the detection of the mRNA target occurs when a PTC is coupled with the splicing machinery . In factus model . In agreus model . In partus model .Nevertheless, several pieces of evidence show that also transcripts not containing an EJC can be targeted by NMD. Indeed, mRNAs without introns can similarly be recognized by NMD and the artificial localization of a proximal intron downstream a physiological stop codon does not affect the stability of the transcript, and the splicing process correctly occurs , 14. TakC. elegans [A prominent role of NMD pathway, in addition to the recognition of nonsense transcripts, is to act as a post-transcriptional regulator of entire groups of genes further indicating that NMD is able to target also not mutated mRNAs as shown in yeasts . In this elegans , finding elegans .The NMD pathway is able to guarantee a fine quality control of transcriptome with a precise timing of expression of NMD targets. This allows cells to adapt in response to stress conditions or modify transcriptome during development. The importance of NMD is evidenced by the fact that the knockout of NMD components, such as Upf1 and Smg1, causes lethality in mice and has been associated with intellectual disability in humans . Indeed,The NMD pathway regulates a set of genes involved in the integrated stress response (ISR) and in the unfolded protein response (UPR), preventing their activation in the presence of low levels of stimuli . On the NMD detects PTCs, which can be caused by frameshift indel mutations, and results in the degradation of mutated mRNAs potentially encoding novel immunogenic peptides. This concept implies that the inhibition of the NMD machinery could increase the production of neoantigens especially in unstable and error-prone cells, such as tumor cells. Accordingly, it has been shown that the genetic inhibition of Upf2 or Smg1 resulted in immune-dependent reduction of tumor growth in syngenic mouse models . On the Along with the effect on the expression of immunogenic peptides, the inhibition of NMD allows the expression of different toxic mutated proteins and results in an anti-tumor effect. The inhibition of NMD could hence represent a strategy to stimulate the re-expression of a plethora of NMD-suppressed genes , 46 to iAs mentioned above, NMD can favor cancer cells by preventing the expression of mutated antigens with immunogenic activity, but it is also endowed with opposite activities with tumor-suppressor functions. For example, mutated BRCA1, which is responsible for breast and ovarian familiar cancer, can display dominant negative activities when prematurely truncated . NonetheIn agreement with an anti-tumor role of NMD, there are several findings showing that this pathway is repressed along with tumor evolution and development. The inhibition is especially triggered by microenvironmental cues as hypoxia  which reNMD is hence inhibited by TME-induced stress to favor tumor adaptation to the surrounding hostile conditions. However, by causing the degradation of a plethora of mutated mRNA and potentially controlling about one third of all alternatively spliced mRNAs , NMD in Since NMD regulates the transcription levels of many pathways in normal and cancer cells, it is easy to deduce that NMD could also impact a plethora of cell responses and ultimately also influence anti-cancer therapy efficacy. Traditional chemotherapy relies on the activation of apoptosis which is negatively regulated by NMD . InteresIn the presence of a dominant nonsense mutation, NMD reduces the expression of the mutated allele having so an important role in reducing the disease phenotypic manifestations . In otheThe regulation of the NMD pathway is extremely variable among the population and the level of degradation of nonsense mRNA, which are the target of readthrough molecules, is patient-dependent , 88. DueDue to the inter-patient different sensitivity to treatment, the association between readthrough agents and NMD inhibitors is focus of several studies that demonstrate a synergistic effect in the rescue of nonsense mutations . DespiteSeveral compounds have been identified as NMD inhibitors, limiting the UPF1 phosphorylation and dephosphorylation cycle . An examThrough a high throughput drug screen, a small molecule, referred to as NMDI-1, was identified and it was shown to be able to stabilize the hyperphosphorylated state of UPF1, so determining NMD inhibition. NMDI-1 was shown to be rather specific and induce low toxicity in mammalian cells, also when used at concentration higher than necessary . In ordewww.clinicaltrials.gov). Amlexanox stabilizes nonsense mRNAs in a late stage of NMD pathway and so acts with a different mechanism from the other analyzed inhibitors [Instead, a repurposing drug approach through virtual screening of FDA-approved drugs allowed the identification of a new promising NMD inhibitor, amlexanox. It is an anti-inflammatory and anti-allergic compound approved for the treatment of mouth ulcers and asthma, and moreover tested for a Phase II clinical trial for diabetes mellitus type II , cells  both in On the other hand, as already mentioned, NMD appears to be endowed with tumor suppressor activity in some tumors. In these settings, an activation of NMD would be preferable. Although a pharmacological approach to restore NMD activity is difficult to design, a possible strategy could be represented by the modulation of the diet. Caloric restriction has already been shown to enhance the efficacy of chemotherapy , hormoneNMD was initially identified as a mechanism to merely prevent the expression of mutated truncated proteins which have the potential to be toxic by displaying dominant negative activities. Later works have clarified that NMD is endowed with a more wide effect on basically every cellular aspect through its capability to control the stability of mutated and non mutated mRNAs. NMD function is also connected to the splicing machinery activity further increasing its capability to control cell physiology and adaptation to stress. NMD has been shown to play a crucial role in tumors although the intimate mechanisms and effects of NMD in cancer cells are still poorly characterized. Many works have proved a pro-tumor effect of NMD, but, at the same time, a large amount of evidence shows that NMD can display a tumor suppressor activity. Undoubtedly, NMD represents an opportunity for new cancer therapy strategies, pending the comprehension of the intricate setting-dependent features. The identification of predictive markers of response to NMD inhibition is hence crucial. An approach could be represented by quantification of NMD activity in cancer cells, which can be higher or lower compared to normal tissues in a patient-dependent fashion .Patients bearing tumors characterized by high levels of NMD activity could theoretically benefit from molecules inhibiting one or more of its mediators. Several compounds are currently been tested for genetic diseases with the final aim of restoring, via NMD inhibition, the expression of mutated proteins that, although truncated, could preserve at least in part their activity and eventually reverting the aberrant phenotype. In cancer settings, NMD inhibition could promote the accumulation of cancer cell-specific neoantigens, potentially immunogenic Fig. . NotablyIn conclusion, NMD may offer a novel opportunity in cancer treatment though it seems to display different and even opposite effects depending on the context. Still, the characterization of this pathway could support the designing of new strategies to treat tumors, especially in combination with immune-based therapies making mandatory the detailing of the intricate mechanisms controlling NMD and the identification of predictive markers which foresee the benefit of NMD inhibition.


Gut maturation during early life involves a series of irreversible, both structural and functional changes that culminate during weaning, when mothers milk is replaced with solid (complex) food. These changes affect the motility, secretion, absorption, and barrier properties of the gastrointestinal tract, nervous and hormonal regulation of gut function and the gut-associated immunity , 2. DespPrevious studies, using newborn animals as models for human infants, imply that gut maturation, culminating naturally at weaning or induced precociously by enteral provocation, for example by polyamines, lectins, or enzymes, is initiated by luminal cues affecting the intestinal epithelium. This activation results in proliferation and maturation of the mucosal layer, the underlying tissue layers, including the gut-associated lymphoid tissue, as well as the accessory gut organs, including the pancreas . CompariThe aim of the Research Topic was to highlight the progression of our knowledge in the regulation of gut maturation and how the process can be induced precociously or delayed through environmental and dietary intervention. Different aspects were addressed in the seven contributions included in the Research Topic, with five original research papers and two reviews.Out of the seven contributions, four papers dealt with the effects of diet on precocious gut maturation.Reicher et al.. They showed that feeding chicks a standard starter diet early, upon hatch, compared to delaying feeding by 1 day, stimulated small intestinal epithelial proliferation and differentiation, which in turn expanded the digestive, absorptive and secretory cell populations during the post-hatch period of 10 days.The effect of early feeding on intestinal maturation in newly-hatched chicks was evaluated in the study by Buddington et al.. The authors found that the effect of protein and leucine on intestinal growth and function, i.e., brush-border carbohydrase activity, was greater for low-weight pigs compared to average-weight pigs. The study also revealed that the pre-term piglet could serve as a relevant model for the identification of formula components that promote gut growth and development in premature human infants.The effect of feeding formulas of differing protein content, as well as the addition of two growth promoters, leucine and hydroxy-methylbutyrate, on organ growth and intestinal function in pre-term pigs was studied by Arevalo Sureda et al. studied the effects of feeding regimen, par-enteral vs. enteral feeding, and feeding diets on exocrine pancreas maturation in both pre-term and full-term piglets. It was found that pancreatic digestive enzymes showed different postnatal developmental patterns and responsiveness to enteral feeding. Trypsin increased with fetal age at birth, amylase increased with postnatal age, while lipase decreased with age. Colostrum inclusion to the diet increased amylase, but decreased trypsin, without any consistent effects on lipase. The low activities of the key enzymes, amylase and trypsin, in pre-term piglets indicate an immature pancreas and reduced digestive capacity. However, rapid enzyme increases appeared after birth following colostrum supplementation. The study confirmed that dietary manipulation also affects the gut accessory organs.Mukonowenzou et al. reviewed recent findings on the impact of dietary medicinal plants and phytochemicals on gut maturation. They addressed different plant products that either directly interact with the gut mucosa, like phytohemagglutinin, or affect the gut microflora producing fermentation products, like SCFA, that indirectly have trophic effects and induce precocious functional maturation. They also reviewed the negative effects of plant products on gut function and maturation. The know-how and use of medicinal plants and phytochemicals, that in many cases are relatively cheap and readily available, being stimulatory of gut maturation may alleviate problems arising in live-stock animals or premature babies with an immature gut.Wang et al. and Słupecka-Ziemilska et al. focused on the effects of maternal dietary composition during gestation and lactation, with either a high-fat diet or one supplemented with probiotics/symbiotics. The effects of feeding rat dams a high-fat diet during gestation and lactation on the intestinal development of their offspring at 3 weeks of age was investigated. The high-fat diet increased the pups body weight and adiposity and resulted in small intestinal hypertrophy. Epithelial brush border enzymes were affected, with decreased sucrase and increased aminopeptidase A activity observed. Intestinal contractility (in vitro) in response to cholinergic stimulation was also affected. The study revealed that maternal consumption of a high-fat diet negatively impacted the structural and functional maturation of the gut in her offspring and may predispose them to metabolic disorders later in life.Wang et al. showed that supplementation of the sow's diet with probiotics (lactobacilli and saccharomyces) or symbiotics (xylo-oligosaccharides) during gestation and lactation improved the intestinal epithelial morphology of the weaned piglets. This diet affected the intestinal microflora balance with increased levels of bifidobacterial and clostridia observed, which were probably associated with the improved intestinal immunity. Thus, maternal interventions with probiotics or symbiotics are an alternative strategy to influence the intestinal microflora of the offspring, improving their gut maturation.Socha-Banasiak et al. focused on one of the current hot scientific topics, that of fetal and neonatal microbial gut colonization and its impact on gut maturation in human infants. The authors bring up the controversial in-utero colonization and the effects of maternal microbiota colonization after birth on acceleration of infant gut development and its benefits on metabolic and immunological function.Related to this, the review by The contributions of this Research Topic have highlighted some initiating cues and mechanisms involved in precocious gut maturation in mammals and birds, including many important livestock animals that in today's intensive production environment are commonly affected by gut-related complications due to immaturity. The knowledge could also be applied translationally to improve strategies for the prevention or treatment of gut-related complications arising in infants, especially those born pre-term, being underweight or formula-fed.All authors contributed equally to manuscript writing, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Diabetic retinal disease (DRD) remains the most common cause of vision loss in adults of working age. Progress on the development of new therapies for DRD has been limited by the complexity of the human eye, which constrains the utility of traditional research techniques, including animal and tissue culture models—a problem shared by those in the field of kidney disease research. By contrast, significant progress in the study of diabetic kidney disease (DKD) has resulted from the successful employment of systems biology approaches. Systems biology is widely used to comprehensively understand complex human diseases through the unbiased integration of genetic, environmental, and phenotypic aspects of the disease with the functional and structural manifestations of the disease. The application of a systems biology approach to DRD may help to clarify the molecular basis of the disease and its progression. Acquiring this type of information might enable the development of personalized treatment approaches, with the goal of discovering new therapies targeted to an individual’s specific DRD pathophysiology and phenotype. Furthermore, recent efforts have revealed shared and distinct pathways and molecular targets of DRD and DKD, highlighting the complex pathophysiology of these diseases and raising the possibility of therapeutics beneficial to both organs. The objective of this review is to survey the current understanding of DRD pathophysiology and to demonstrate the investigative approaches currently applied to DKD that could promote a more thorough understanding of the structure, function, and progression of DRD. Diabetic Retinal Disease (DRD) is a complication of diabetes responsible for significant morbidity and decreased productivity and quality of life ,2. The gThe current standard for DRD diagnosis relies on the decades-old Early Treatment Diabetic Retinopathy Study (ETDRS) grading scale based exclusively on vascular abnormalities ,38,39,40Psychophysical tests, a series of well-validated assessments, have found a subset of patients to have deficits in peripheral vision, night vision, color-hue discrimination, and contrast discrimination during the preclinical DR stage, before the development of vascular abnormalities ,47,48,49Like DRD, DKD has historically been viewed as a vasculopathy responsible for significant morbidity and mortality in diabetes. Indeed, DKD is consistently the most common cause of end-stage kidney disease (ESKD) in the United States ,66. A reHowever, this classic DKD description does not capture the wider clinical variability of this disease. While some studies have estimated microalbuminuria to have an 80% predictive value for DKD progression ,68, neweThis variation in DKD and DRD presentations and the existence of multiple phenotypes complicates our ability to understand, predict, and treat this disease, necessitating novel research approaches to determine the basis for this complexity. Such efforts are paramount in order to advance the current DKD treatment paradigm, which largely remains unchanged since the discovery of angiotensin receptor inhibition in the 1990s and consists of glycemic control, blood pressure management, fluid balance, and angiotensin-converting enzyme (ACE) inhibition ,75,76,77In contrast to reductionist investigations that focus on single or a few molecules, the power of systems biology lies in its unbiased and agnostic multi-scalar integration of data generated from genomics, transcriptomics, proteomics, metabolomics, and lipidomics studies, along with tissue morphometry and clinical data and biomarkers to interrogate the true physiologic state of individuals in health and disease  Figure . SystemsSystems biology studies have identified trackable biomarkers that correlate closely with structural and functional abnormalities early in DKD, including urine haptoglobin , urinaryIn addition to these advances to diagnostics , systemsTogether, the recent progress in DKD research highlights a new framework by which advances in personalized diagnostics and novel therapeutics can intervene at a time point early enough when disease progression is reversible. This approach is in contrast to DRD clinical phenotyping modalities, for which obtaining biosamples for systems biology analyses is not part of clinical care . One pot“Diabetic renal-retinal syndrome” describes the clinical phenomena of: (1) DRD vascular abnormalities as predictors of impaired renal function ,88; and Indeed, an important step forward in accurately assessing DKD and DRD lies in determining the spectrum of disease phenotypes ,58,59,70The ultimate manifestation of intimate collaboration is the development of novel therapeutics for the complications of diabetes. Historically, the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC), UK Prospective Diabetes Study (UKPDS), and Action to Control Cardiovascular Risk in Diabetes (ACCORD) trials established the importance of glycemic, blood pressure, and dyslipidemia control in DRD therapy ,103,104.However, systems biology-driven insights offer an alternative approach to therapeutics development. One example is the aforementioned JAK-STAT pathway affected in DKD. As the JAK-STAT pathway is also affected in DRD , it is pRegardless of whether baricitinib-mediated suppression of albuminuria  translatAs these systems biology studies integrate the various levels of data , it becoThe fields of DRD and DKD have progressed dramatically since their categorizations as complications of diabetes. With the use of systems biology, new signaling pathways, biomarkers, and therapeutic targets have been identified for both diseases. However, there continues to exist a need to integrate these findings into the clinical evaluation and treatment of DRD and DKD. By allowing these two diseases to walk in tandem with multi-scalar investigation and assessment scientifically and medically, new strategies effective in the care of diabetic patients may be revealed.Progress in DRD research and new therapies has been exceedingly slow with the reliance on classic reductionist scientific methods. Given the recent boon in development of systems biology techniques applied to other complex diseases such as DKD, DRD researchers can now take advantage of these transformative technologies. By employing agnostic analytic methods to big datasets, DRD research has an unparalleled and unbridled opportunity to reveal novel pathomechanisms involved in human DRD. Armed with more specific knowledge of human pathomechanisms of DRD, researchers will then be able to reapproach model systems to determine which model most closely recapitulates human biology of the research question to be asked . FurtherTowards this goal, recent progress in human stem-cell-derived retinal organoids  provides


To the EditorBased on our extensive clinical experience, we are very concerned about the choice of antihypertensive drugs in the elderly. Recently, an authoritative guide recommended the use of thiazide diuretics, calcium channel blockers (CCB), angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB) for hypertension, and thiazide diuretics as the first-line therapy in the majority of elderly hypertensive patients. There is evidence that the use of thiazide diuretics reduces comprehensive cardiovascular outcomes in hypertensive patients over 65 years of age, and thiazide diuretics  are better than other categories of drugs for older hypertensive patients . Based oTherefore, although we agree with the application of diuretics in the treatment of hypertension in the elderly, the concurrent diseases of elderly patients must be considered and the risk of possible adverse events after antihypertensive treatment must be carefully analyzed. Furthermore, the mode of administration of diuretics is also worth considering. For example, a study recommended to take antihypertensive drug once a day in the afternoon, which will help in controlling blood pressure, reducing water and sodium retention, and nocturia, thereby reducing the risk of adverse events .


Numerous studies have indicated that the progression of chronic kidney disease (CKD) to end-stage renal disease (ESRD) is strictly associated with the accumulation of toxic metabolites in blood and other metabolic compartments. This accumulation was suggested to be related to enhanced generation of toxins from the dysbiotic microbiome accompanied by their reduced elimination by impaired kidneys. Intestinal microbiota play a key role in the accumulation of uremic toxins due to the fact that numerous uremic solutes are generated in the process of protein fermentation by colonic microbiota. Some disease states, including CKD, are associated with the presence of dysbiosis, which can be defined as an “imbalanced intestinal microbial community with quantitative and qualitative changes in the composition and metabolic activities of the gut microbiota”. The results of studies have confirmed the altered composition and functions of gut microbial community in chronic kidney disease. In the course of CKD protein-bound uremic toxins, including indoxyl sulfate, p-cresyl glucuronide, p-cresyl sulfate and indole-3-acetic acid are progressively accumulated. The presence of chronic kidney disease may be accompanied by the development of intestinal inflammation and epithelial barrier impairment leading to hastened systemic translocation of bacterial-derived uremic toxins and consequent oxidative stress injury to the kidney, cardiovascular and endocrine systems. These findings offer new therapeutic possibilities for the management of uremia, inflammation and kidney disease progression and the prevention of adverse outcomes in CKD patients. It seems that dietary interventions comprising prebiotics, probiotics, and synbiotics could pose a promising strategy in the management of uremic toxins in CKD. Some disease states, including chronic kidney failure, chronic kidney disease (CKD), obesity, diabetes, cardiovascular disease, inflammatory bowel disease, and cancers are associated with the presence of dysbiosis, which can be defined as an “imbalanced intestinal microbial community with quantitative and qualitative changes in the composition and metabolic activities of the gut microbiota” ,2,3,4. TThe intestinal dysbiosis resulting from the loss of kidney function is associated with the secretion of urea into the gastrointestinal tract and successive hydrolysis of urea by some gut microbes . ConsequCKD-related alterations of gut microbial community were also suggested to be related to dietary restrictions aiming at the prevention of hyperkalemia as well as and malnutrition. Moreover, patients with CKD are frequently polymedicated . The freHowever, the question of the exact mechanisms related to gut microbiota-mediated changes in ESRD metabolome remains unanswered . The unrOver 100 trillion microbial cells are present in the human gut constituting the gut microbiota . The resThe intestinal epithelium, which is a single layer of columnar epithelial cells, plays a vital role in nutrient absorption, as well as acting as a natural barrier preventing or hampering systemic translocation of pathogens and antigens . IntestiGut microbiota is involved in multiple functions, for example the synthesis of certain vitamins (K and B groups), the breakdown of indigestible plant polysaccharides, the activation of bioactive food components , the degradation of dietary oxalates and the biotransformation of conjugated bile acids, which contributes to the nutritional balance ,39,40,41In the course of CKD, microbiome composition and the intestinal environment undergo transformation from a symbiotic to a dysbiotic state which is mirrored by an increase in colonic protein fermentation and resulting rise in microbiota-derived uremic toxins as well as a decrease in carbohydrate fermentation and subsequent diminished formation of host-beneficial metabolites  ,6. AccorThe presence of dysbiotic gut microbiome is not only associated with enhanced production of harmful metabolites and uremic toxins, but also with the cessation of beneficial metabolites  formation. Wong et al.  reportedTherefore, it seems that in general, CKD-related alterations upregulate microbiota producing specific uremic toxins and downregulate microbiota generating beneficial products . This thThe development of gut microbiome dysbiosis in CKD patients could result in impaired protein assimilation, prolonged intestinal transit time, intestinal wall edema, metabolic acidosis, iron therapy and diminished consumption of dietary fiber ,59,60,61In normal conditions, uremic toxins which bind with plasma albumin are excreted into urine via by organic anion and cation transporters present in the kidney tubular cells (tubular secretion) which seems to be the predominating mechanism ,94. In tReduced fractional kidney clearance implies that tubular clearance is more disturbed than glomerular filtration. Therefore, it is not surprising that the highest levels of uremic toxins are found in end-stage renal disease undergoing dialysis .Indole is an intercellular signal molecule which modulates diverse biological functions; for example it upregulates gene expression of gut epithelial cell junctions and controls expression of pro- and anti-inflammatory factors in intestinal epithelial cells therefore enabling the maintenance of host-microbe homeostasis at the mucosal surface ,100,101.p < 0.05) [Phenols are other aromatic compounds which are produced by intestinal bacteria  via partial breakdown of tyrosine and phenylalanine . The uri < 0.05) .Rossi et al.  reportedApart from the aforementioned, also other toxins  also come from bacterial metabolism ,82,131. Finally, TMAO (molecular weight 75 kDa) is a gut-derived toxic metabolite obtained as a product of bacterial metabolism of quaternary amines . Trimethylamine formed in the first step is later absorbed and converted to TMAO by flavin monooxygenase enzymes in the liver . TrimethIt has been demonstrated that dialysis patients may suffer from the toxin-induced residual uraemic syndrome, severely affecting the quality of their lives, due to the fact that traditional renal replacement therapy does not efficiently remove these toxins . Some auThe diagnosis as well as the treatment and prevention of some diseases requires the knowledge of the composition, dynamics, and stability of gut microbiota . NumerouProbiotics contain i.e., living species of Bifidobacteria, lactobacilli, and streptococci which are capable of altering gut microbiota and soothing the inflammatory state ,153. TheNumerous studies have indicated that also the intake of prebiotics, which are non-digestible food ingredients stimulating the growth or activity of bacteria in the colon, may also significantly decrease the generation of uremic toxins. Prebiotics stimulate the growth of Bifidobacteria and Lactobacilli species at the expense of other groups of bacteria in the gut . The conThe use of synbiotics  was proved to be beneficial in CKD. A trial investigating the combination of probiotic and prebiotic therapies for over six weeks in pre-dialysis CKD patients found that such therapy lowered serum p-cresyl sulphate and gut microbiome alterations, but it did not diminish considerably serum IS . The redRecently, the knowledge of the metabolic potential of gut microbiome and its vital role in the pathogenesis of several chronic inflammatory diseases has widened rapidly. The gut seems highly attractive as a future target for attenuating uremia-related complications. However, there is still a need for subsequent studies focusing on the confirmation of gut microbiome pattern in kidney diseases and the analysis of associations between different types of kidney diseases and the gut microbiome. It has already been demonstrated that the presence of chronic kidney disease may be accompanied by the development of intestinal inflammation and epithelial barrier impairment leading to hastened systemic translocation of the bacterial-derived uremic toxins  and consequent oxidative stress injury to the kidney as well as to cardiovascular and endocrine systems. These findings have provided new therapeutic possibilities for the management of uremia, inflammation and kidney disease progression and the prevention of adverse outcomes in CKD patients. Numerous interventions aiming to restore proper gut microbiota composition and to slow the progression of kidney diseases have been examined. It seems that dietary interventions comprising prebiotics, probiotics, and synbiotics could pose a promising strategy in the management of uremic toxins in CKD. However, due to great variability of studies (in terms of the type used and the dose of fiber/prebiotic/probiotic/synbiotic) conducted so far, as well as the small number of trials’ participants, short follow-up and/or lack of dietary control make the formulation of final conclusions is impossible. It seems that more detailed research focused on the gut as a potential source of CKD-related complication may bring interesting discoveries. Despite the lack of conclusive results concerning the beneficial impact of the aforementioned compounds, a healthy dietary pattern comprising large amounts of whole fruits, grains, and vegetables should be encouraged in CKD patients due to their confirmed advantageous effect beyond uremic toxin production. However, it should be kept in mind that some fruits and vegetables contain high potassium levels and therefore, their consumption must be limited for many patients with advanced CKD or ESRD. It seems that isolated intervention involving just one type of food may not be effective in restoring proper gut microbial composition. The restoration of functionality and composition of microbiota in CKD patients requires the use of combined measures acting synergistically. Now, in the age of personalized medicine, nutritional interventions should be based on individual microbiota characteristics as well as the prevalence of comorbidities due to the fact that gut microbiota is specific to each person, and comorbidities may alter health requirements of patients. However, also these fields also require an extensive research.


The use of immunosuppressive drugs, broad-spectrum antimicrobials, anticancer chemotherapies, organ transplants, an aging patient population, and the intervention of hospitalized patients with numerous medical devices contribute to compromised immunity, creating an opportunistic possibility for disease by medically important fungi. Fungal infections represent a public health problem due to the synergism of the aforementioned factors and the ability of the fungus to adapt to new environmental conditions, which depends on the expression of specific virulence factors. For instance, the ability to produce specific hydrolytic enzymes, the overexpression of genes that regulate the efflux pumps activity increasing tolerance to antifungal drugs, the ability to alter cell morphology, the production of adhesins facilitating their attachment to surfaces, and the capability of forming biofilms in living tissues or inanimate substrates , are some structural or molecular factors that enhance the pathogenic potential of fungi.Candida spp., Trischosporon spp., Aspergillus spp., Paracoccidioides brasiliensis, and Fusarium spp., have been identified by emerging technologies, as fungal pathogens.Biofilms are structures formed by microbial communities that develop amidst a matrix constituted by extracellular substances, where groups of microorganisms arranged in microcolonies show high resistance to chemical disinfection, antimicrobial therapy, and a high evasion capacity against immune system response. These microbial communities can also serve as protection for the development, nutrition, metabolic cooperation, and acquisition of new genetic characteristics by the participating microorganisms, generating clinical, therapeutic, and economic implications. Among the broad range of yeast and filamentous fungi that form biofilms, From the cell’s attachment to the development of a three-dimensional structure, the growth of a biofilm is correlated with molecular aspects such as transcriptional regulatory genes that contribute to cell differentiation and adaptation to environmental cues. However, classifying the roles of all these genes is a highly complex task since the regulatory processes of biofilm formation are cyclical and dynamic.Disease-related biofilms can be multi-species or even multi-kingdom. It has been suggested that hyphae of filamentous fungi or the filament form (pseudohyphae) of yeast provide the architecture for adhesion and development of bacterial biofilm, allowing the formation of polymicrobial biofilms. These biofilms represent a problem of significant clinical relevance since they a reserve for a wide variety of microorganisms, including fungi and bacteria. The development of polymicrobial biofilms can modify the virulence of microorganisms that are part of these interkingdom communities and the standardization of treatments that are usually effective against these pathogens, can be altered.Due to the limited therapeutic arsenal available to treat systemic fungal infections associated with the increase in antifungal resistance and the number of infections caused by biofilms, the search for new molecules with antifungal potential is necessary. Antifungal drugs for clinical use currently comprise polyenes, flucytosine, azoles, and echinocandins, which, although effective, have a limited activity spectrum, a small number of targets, undesirable side effects, and the emergence of resistant strains. Such factors have motivated researchers around the world to develop new antifungal agents employing synthetic and semi-synthetic methodology, in addition to the isolation of a natural product with antifungal activity. Nevertheless, the emergence of resistance and the low activity of some drugs correlated to their delivery, especially in biofilms, makes it necessary to explore new strategies to develop more effective therapies.The ability to form biofilms on both biotic and abiotic surfaces and increased tolerance and resistance to antimicrobials, including disinfectants, has contributed to the biofilm’s persistence, including fungal biofilms, in the clinical setting. Therefore, effective decontamination of these environments can significantly increase patient safety. Studies have evaluated the efficacy and compatibility with diverse applications of several clinically used disinfectants against fungal pathogens to address these challenges. However, it is difficult to develop efficacious biofilm disinfection strategies, due to the diversity of species found in multispecies communities, broad spectrum microbial susceptibility, and lack of penetration. They must also be environmentally friendly and have low toxicity to humans. Furthermore, a biphasic death pattern may appear when treated with an increasing amount of drugs whereby most of the biofilm population is killed, while a small fraction survives - persister cells. Indeed, if persister cells are isolated, regrown and repeatedly treated with high drug concentrations, the same drug response will be observed as in the original population. Once antimicrobial treatment ends, persistent ones can survive and repopulate the biofilm, being correlated with recalcitrant infections. In fact, biofilm-related infections are increasingly related to high rates of morbidity and mortality in the healthcare system and, therefore, many research efforts are needed to help with its prevention and management. In this Research Topic, a multidisciplinary team of authors who share a common interest in human fungal pathogenesis was brought together to highlight the fungal biofilm-related infections by different fungi through seven original articles and one brief communication.Bitencourt et al., which emphasize the role of the APSES transcription factors family, especially StuA, in the Trichophyton rubrum morphogenesis and virulence. Deletion of StuA impairs biofilm formation by this dermatophyte.The vital role of transcriptional factors in stages of biofilm formation is exemplified by the original research presented by Ramírez-Granillo et al., who analyzed the aspects of the co-infection of Aspergillus fumigatus and Staphylococcus aureus, using a cell line culture model to study the impact of polymicrobial biofilms on keratitis. The complexity of fungal-bacterial interactions in polymicrobial/inter-kingdom biofilms involving dimorphic fungus was highlighted by Medina-Alarcón et al. who showed the pioneering interaction between P. brasiliensis and Mycobacterium tuberculosis as well as the possibility of treating the infection with a nanoparticle-releasing natural compound.The polymicrobial nature of biofilms is recognized as an essential contributor to fungal pathogenesis. This important topic was addressed in the original research by Cymbopogon flexuosus essential oil and its bioactive constituent - citral) on bacterial (Staphylococcus aureus), fungal (Candida species), and mixed bacterial-fungal biofilms was provided by Gao et al. In addition, a pharmaceutical combination of the antifungal compound and innovative approaches has been used to combat fungal diseases, especially those associated with biofilms. Contribution to new antifungal biofilms strategies, combining antifungal compounds and photodynamic therapy, was provided by the original research authored by Bila et al. The antimicrobial delivery systems, a promising tool for the inactivation of fungi in biofilms, were shown by Costa-Orlandi et al. when demonstrating the comparable efficacy of nitric oxide-releasing nanoparticles on dermatophyte biofilms.However, for successful management of fungal infections, the continued development of new antifungals and the appropriate use of commonly used antifungals are necessary. Evidence of the action of plant-derived products , host-pathogen interactions, transcription factors involved in biofilm formation regulation, environmental conditions, and new antifungal drug candidates, and provide the current state-of-the-art landscape in our understanding of biofilms by medically important fungi and relevant bacterial pathogens and warrants future studies to understand the basis of fungal biofilms biology in infection and disease.All authors contributed to the final format of the manuscript and approved it for publication.National Institute of Allergy and Infectious Diseases , São Paulo Research Foundation - FAPESP  and Brazilian National Research Council – CNPq . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.