Online health forums provide rich and untapped real-time data on population health. Through novel data extraction and natural language processing (NLP) techniques, we characterise the evolution of mental and physical health concerns relating to the COVID-19 pandemic among online health forum users.We obtained data from three leading online health forums: HealthBoards, Inspire and HealthUnlocked, from the period 1 January 2020 to 31 May 2020. Using NLP, we analysed the content of posts related to COVID-19.(1) Proportion of forum posts containing COVID-19 keywords; (2) proportion of forum users making their very first post about COVID-19; (3) proportion of COVID-19-related posts containing content related to physical and mental health comorbidities.Data from 739 434 posts created by 53 134 unique users were analysed. A total of 35 581 posts (4.8%) contained a COVID-19 keyword. Posts discussing COVID-19 and related comorbid disorders spiked in early March to mid-March around the time of global implementation of lockdowns prompting a large number of users to post on online health forums for the first time. Over a quarter of COVID-19-related thread titles mentioned a physical or mental health comorbidity.We demonstrate that it is feasible to characterise the content of online health forum user posts regarding COVID-19 and measure changes over time. The pandemic and corresponding public response has had a significant impact on posters’ queries regarding mental health. Social media data sources such as online health forums can be harnessed to strengthen population-level mental health surveillance. This study uses a novel approach to examine the content of a large, unstructured online health forum dataset using natural language processing.Online health forums provide a unique resource of real-world data that represent the lived experiences of people with mental and physical health disorders.It is not yet possible to establish COVID-19 status or whether concerned posters had pre-existing mental or physical health issues, had recovered or became unwell for the first time during the study period.Online health forums are help-seeking forums, which could contribute towards self-selection bias.Measures to tackle the COVID-19 pandemic have resulted in unprecedented societal restrictions worldwide. The mental health impacts of these measures and accompanying socioeconomic stressors are likely to be extensive; identifying and quantifying these impacts are now an urgent priority.2 3Real-world data from online resources may be extracted using natural language processing (NLP) techniques to provide automated, population-level health surveillance. These methods can be used to rapidly ascertain discussion related to COVID-19 and associated symptoms and comorbidities. NLP has previously been used to identify medically relevant information from web pages and analyse extracted text.9While social media platforms such as Twitter, Facebook and Reddit are commonly used, other internet resources such as online health forums have so far been neglected. Online health forums are enriched for health information and receive millions of posts each year, therefore providing untapped reservoirs of healthcare data at population level. Users of online health forums may post information or questions related to a particular health problem and discuss these in written threads with other forum users. Some users are ‘peer experts’ characterised as people with expertise in a particular area of health through lived experience. They may frequently respond to other users’ questions and provide practical insights into managing a particular healthcare problem.11In a recent proof-of-concept study, we demonstrated that online health forums can be extracted to detect health discussion trends that correlate with real-life events.We obtained data from online health forums using NLP. Online forums are discussion websites hosted on the internet where people hold conversations in the form of posted messages. A single conversation is called a thread. Threads are chains of posts identified within a forum by a title and an individual URL. Clicking on the thread title opens the thread which contains one or more posts which may be from the same user who started the thread  or different users who have replied within the thread. In this study, we analysed text data in thread titles and in individual posts within a thread. We analysed posts written in English only. Depending on the forum’s settings, users can be anonymous or have to register with the forum to post messages, with most users opting not to use personally identifiable information to register their account. Registration may not be required for read-only access. Most forums recommend that users do not use personally identifiable information when posting. Online health forums specifically cover health topics and offer peer support for various health conditions.www.healthboards.com), Inspire (www.inspire.com) and HealthUnlocked . These forums were chosen on the basis that they have global user coverage, include subforums on several aspects of healthcare, have a large user base contributing to regular activity on the forum and are feasible to extract information from using NLP.We collected data from three major online health forums posted from 1 January 2020 to 31 May 2020: HealthBoards  medical treatment in an intensive care unit or (2) physical symptoms as a direct consequence of COVID-19 infection or (3) mental health symptoms as a consequence of measures in response to the pandemic.Search terms used to identify whether a thread or post was related to COVID-19 were ‘COVID-19’, ‘COVID-19’, ‘coronavirus’, ‘corona’, ‘sars-cov-2’, ‘sars-2’, ‘shielding’, ‘pandemic*’, ‘vulnerable’, ‘quarantine’, ‘lockdown’, ‘distancing’, ‘isolation’, ‘isolating’ where * indicates a wildcard search term.Data obtained from different online health forums come in various formats. We standardised and normalised the data before analysing them. This included normalisation of Unicode strings and whitespace characters, standardisation of date and time, and standardisation of location through the GeoNames.org database.We identified the users contributing to a COVID-19-related thread in a given week. We then retrieved all the other posts made by the same authors in the previous, same and subsequent calendar weeks. We scanned such posts for physical symptom, mental health symptom or intensive care keywords as defined in We inspected thread titles to identify how many mentioned a comorbidity in the title. We searched for terms related to autoimmune disorders, mental disorders or worry, cancer, cardiovascular problems or stroke, and diabetes as listed in We analysed the first ever post published by a user to determine the proportion of first-time posters who started out by contributing to a COVID-19-related thread. We performed this analysis to determine the degree to which new users were motivated to make their first post in relation to the COVID-19 pandemic and how this varied over time during 2020.All descriptive analyses were performed using bespoke software written in Python. An outline of the coding approach employed is included in the We consulted and adhered to internet research guidelines from the Association of Internet ResearchersGiven licensing and privacy issues, it is not possible to publicly release the aggregate dataset generated from the three online health forums investigated. However, we welcome collaboration with other researchers and healthcare policy makers. Anyone interested in accessing the aggregate data and data analysis code should contact the guarantor .Patients and the public were not involved in the design or conduct of the study.HealthUnlocked was the most frequently used forum accounting for 97% of overall posts and 97% of posts mentioning COVID-19 in the thread title or post content during the study period .Weekly post count for HealthUnlocked peaked in mid-March. Post count for Inspire declined sharply in the last 2 weeks of March. Post count for HealthBoards declined slowly across the entirety of the observation period .Across all three forums, there were a total of 3342 threads containing a COVID-19 keyword within the thread title or URL. These contained a total of 44 894 posts during the study period (1 January 2020 to 31 May 2020). A total of 35 581 posts (whether in COVID-19-related threads or otherwise) contained a COVID-19 keyword during the study period. The proportion of posts containing COVID-19 keywords increased rapidly across all forums in early March , correspFor quite a long period, most posts about COVID-19 (over 90% at the beginning of the observation period and remaining above 50% until the week ending 29 March) were written by users who had not yet posted on the topic. By the end of the observation period, the percentage of weekly posts in COVID-19 threads written by new entrants to the discussion reduced to a still quite sizeable 30%. While many of these users may have posted before on the forum about other topics, Over a quarter of COVID-19-related thread titles mentioned another condition of interest . After cPosts in threads related to COVID-19 were analysed to determine the number of users contributing in each given week. For each active user, all posts in the previous, same and following calendar weeks were scanned irrespective of thread for mentions of physical symptom, mental health symptom or intensive care keywords. The number of active users mentioning each of these concerns peaked in the week ending 22 March and subsequently declined but still remained elevated above the January baseline. In particular, users discussing mental health outnumbered users mentioning the other topics .Using a novel technique to analyse data from online health forums, we found a marked increase in posts related to COVID-19 across the observation period of 1 January 2020 to 31 May 2020. The frequency of these posts increased rapidly in early March 2020 corresponding with the WHO’s declaration of COVID-19 as a pandemic.During this period, we found mental health symptom keywords were most frequently mentioned by authors of COVID-19-related posts , followed by physical symptoms and intensive care keywords, suggesting that the pandemic and public health response to it has had a significant impact on posters’ concerns regarding mental health. The marked increase in mental health symptom-related posts in early March, when the WHO declared the COVID-19 pandemic, correlates with preliminary worldwide data that show increases in anxiety and depression in response to the outbreak.The mental health impacts of COVID-19 and associated physical distancing restrictions are likely to be extensive and wide-reaching. There is a growing body of evidence supporting the neuropsychiatric effects of coronavirus infections.17A preliminary survey of 3545 German respondents found evidence of substantial mental health burden from travel and physical distancing restrictions, including increased levels of stress, anxiety, depressive symptoms, sleep disturbance and irritability.20Our findings also suggest that mental and physical health concerns documented in online forum posts have levelled off following their peak in March 2020 but their rates continued to remain above pre-pandemic levels. The number of users active in COVID-19 threads who also wrote posts concerning mental health symptoms reduced from their peak in March of 1355 (per week) to 253 by the end of the observation period (compared with a mean number of 30 per week in January), suggesting that as time went on most users had begun to adjust to the consequences of the pandemic. Furthermore, the increased engagement of users on online health forums could be related to the restrictions on travel and in-person interaction during the pandemic, leading to a shift to social engagement through online media. Other NLP studies have also identified a similar trend. An analysis of 10 million Google searches within the USA found large shifts in mental health symptom searches linked to stay-at-home orders in the USA across the week commencing 16 March 2020.We used keywords related to intensive care treatment in NLP models reflecting the potential for COVID-19 to lead to serious illness requiring intensive care treatment. We found that threads containing content related to intensive care treatment increased in frequency during the pandemic. Follow-up data in more recent studies has highlighted the potential for COVID-19 to be associated with prolonged, multisystem involvement associated with significant disability,Over the entire period, on average 4% of first-time posters (over 20% in the peak period) made their very first contribution to the forum in a COVID-19-related thread. Furthermore, 77% of COVID-19 threads were started by users who had never posted about the topic before and chose to start out by creating their own thread. A certain degree of motivation is required to take someone to the point of making that first post on a forum and also for starting a thread; our finding suggests that the pandemic is driving users to engage more actively in community forum services in times of uncertainty.Online health forums are an important source of real-world, real-time, population-level data on people living through the COVID-19 pandemic. Online health forums also afford users anonymity to discuss aspects of their experience they might otherwise have been embarrassed or fearful to disclose in identifiable forms of social media. We have demonstrated that it is possible to automate information extraction from these posts using NLP, providing access to a rich reservoir of previously untapped real-world data from health-specific online resources.Our approach was able to automatically extract data from a large sample of over 53 000 unique users at a fraction of the cost of previous approaches that have relied on social media individual participant recruitment and manual review of posts generating sample sizes in the low hundreds.8Our study has some limitations. At present, it is difficult to establish whether concerned posters have pre-existing mental or physical health issues, have experienced confirmed COVID-19 illness themselves, are recovered or have become unwell for the first time. Online health forums are help-seeking communities; this introduces self-selection bias in which individuals from disadvantaged backgrounds who do not have IT equipment/network connection to access online resources are under-represented and our results are therefore not generalisable to the entire population. Furthermore, as these forums have worldwide coverage, we cannot isolate trends to one geographic region. However, future work could use the location data (see Data pre-processing in Methods) to explore this avenue. We analysed data up to 31 May 2020 providing a relatively short period of follow-up following the onset of the COVID-19 pandemic. Future studies examining a longer period of follow-up would help to determine if the increase in activity and content related to COVID-19 and comorbid health problems persisted. As we analysed anonymised data, we were unable to investigate the demographic characteristics of the forum users in our study to determine whether they were representative of the wider population.Publicly accessible sources of real-world data, such as online health forums analysed in this study, can strengthen population-level physical and mental health surveillance and provide a rapid and inexpensive means to inform public healthcare policy. We found that the majority of posts in online forum data related to COVID-19 concerned features related to mental health and that the peak in frequency of posts corresponded with the early phase of the pandemic, indicating the significant impact of COVID-19 on the mental health of susceptible populations.As the pandemic evolves, further research using online forum data could improve our understanding of the long-term consequences of COVID-19 infection


African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family. Despite its agricultural importance, little is known about the fundamental molecular mechanisms of this pathogen. Short-read sequencing (SRS) can produce a huge amount of high-precision sequencing reads for transcriptomic profiling, but it is inefficient for comprehensively annotating transcriptomes. Long-read sequencing (LRS) can overcome some of SRS’s limitations, but it also has drawbacks, such as low-coverage and high error rate. The limitations of the two approaches can be surmounted by the combined use of these techniques. In this study, we used Illumina SRS and Oxford Nanopore Technologies LRS platforms with multiple library preparation methods (amplified and direct cDNA sequencings and native RNA sequencing) for constructing the ASFV transcriptomic atlas. This work identified many novel transcripts and transcript isoforms and annotated the precise termini of previously described RNAs. This study identified a novel species of ASFV transcripts, the replication origin-associated RNAs. Additionally, we discovered several nested genes embedded into larger canonical genes. In contrast to the current view that the ASFV transcripts are monocistronic, we detected a significant extent of polycistronism, although a large proportion of these transcripts are expressed in low abundance. A multifaceted meshwork of transcriptional overlaps was also discovered. High-throughput massively parallel sequencing has revolutionized modern biology by facilitating the explosive growth of genomics and transcriptomics. Next-generation short-read sequencing (SRS) has allowed producing a tremendous volume of data with unprecedented speed. Currently, Illumina rules the SRS market. RNA studies are hampered by the limitations of SRS that cannot sufficiently cope with the complexity of large transcriptomes . Third-gOrnithodoros, which makes ASFV the only DNA virus that can infect both mammals and arthropods [African swine fever (ASF) is a highly lethal hemorrhagic disease of domestic pigs. Its causative agent is the ASF virus (ASFV) that is the sole member of the Asfarviridae family . The natthropods . To datethropods . GenotypASFV belongs to nucleocytoplasmic large DNA viruses. Even though some form of nuclear replication is detected in the early phase of the infection , it mainWith a few exceptions , most ofMolecular biological analysis of ASFV is not easy because most wild-type viral strains cannot be propagated in established cell lines . The virThe transcription kinetics of ASFV has only been studied in detail in a few cases. The temporal regulation of ASFV gene expression appears to be similar to the transcription cascade described in poxviruses ,23, in wThe main objective of this study is to gather additional information about the transcriptomic architecture of a wild-type ASFV in natural host cells by applying a dual sequencing multi-technique approach. We cataloged all types of the transcript, including mRNAs, non-coding RNAs (ncRNAs) and transcript isoforms, and carried out a semiquantitative analysis of the expression levels of the transcripts at different stages of viral infection. Clarifying the functional elements of the viral genome is vital for a better understanding of the unique biology of this dangerous pathogen.The experimental design utilized in this study is visualized in 2 in air gas phase. The infectious titer of serially diluted viral stock was calculated using an immunofluorescence (IF) assay as described earlier [5 cells and infected at a multiplicity of infection (MOI) of 10 plaque-forming units per cell at 4 h after cell seeding. The supernatant was replaced with a fresh medium after 1 h post-infection (hpi). Infected PAM cells were harvested at 4, 8, 12, and 20 hpi, whereas mock-infected cells were harvested at 20 hpi IF assay was used for monitoring the efficiency of infection in an infected control well fixed at 20 hpi.Porcine alveolar macrophage (PAM) cells were freshly harvested from swine lungs according to the OIE Manual’s instructions . PAM cel earlier . PAMs weThe length of the ASFV infection cycle length is approximately 18–22 h ,28, yet ® RNA  kit for isolation of the total RNA from the samples as was described in our previous publications [We used the NucleoSpinications . BrieflyFor the direct RNA (dRNA) and direct cDNA (dcDNA) sequencing approaches, the poly(A)+ fraction of the total RNAs were extracted. This process was carried out with the Qiagen’s Oligotex mRNA mini kit using spin columns according to the kit’s manual.® rapid directional RNA-Seq Kit 2.0 was used for the preparation of strand-specific single-end or paired-end RNA libraries as recommended by the manufacturer. The following steps were used: RNA fragmentation, first-strand synthesis, second-strand synthesis, adenylation, adapter ligation, and PCR amplification. The ribodepletion was carried out according to the kit’s instructions.The RiboMinus™ Eukaryote System v2 (Thermo Fisher Scientific) was used to obtain rRNA-free RNA samples, which is required by the applied Illumina library preparation approach. For explanation, the NEXTFLEXNative RNA sequencing was carried out using the ONT SQK-RNA002 kit as described earlier ,34. In bThe same polyA (+) RNA mixture was used for the preparation of the dcDNA library as for dRNA sequencing. The library was generated with the ONT’s direct cDNA sequencing kit (SQK-DCS109) according to ONT’s protocol. Briefly, the reverse transcription (RT) reaction was conducted in the presence of anchored oligo(dT) primers (VN primer from the ONT Kit), dNTPs, buffer and enzyme from the Maxima H minus reverse transcriptase kit (Thermo Fisher Scientific), as well as strand switching primer . To avoid RNA degradation, RNaseOUT™ (Thermo Fisher Scientific) was also added to the reaction. After cDNA synthesis, the RNA strands were digested using an RNase cocktail enzyme mix (Thermo Fisher Scientific). The sample was cleaned using the XP bead purification method with AMPure XP beads (0.8 BR) (Beckman Coulter). Samples were incubated for 5 min on the HulaMixer; then, they were cleaned as was described in the dRNA-seq section. The second cDNA strand was generated using LongAmp Taq master mix from New England Biolabs and the PNT’s PR2 primer. Double-stranded cDNAs were washed with XP beads then were handled with NEBNext Ultra II end-prep enzyme mix (New England Biolabs). After an XP bead-washing, the sample is ligated to the ONT adapter mix using blunt/TA ligation master mix (New England Biolabs). Finally, 200 fmol from the XP bead-purified sample was run on two SpotON Flow Cells.Total RNA samples from each time point  were sequenced individually using the ONT MinION device and the cDNA-PCR Barcoding protocol (SQK-PCS109 and SQK-PBK004). RT was carried out as described in the dcDNA protocol (above). The samples were amplified using the LongAmp Taq master mix. Low-input barcode primers (ONT’s SQK-PBK004 kit) were added to the samples with the aim of multiplexing the samples on the Flow Cells. After the PCR reaction, cDNAs were treated with an exonuclease, and they were finally cleaned with AMPure XP beads, as it was explained in the dcDNA paragraph.® rapid directional qRNA-Seq kit  was used for library preparation from the rRNA-depleted sample. All reagents and enzymes were supplied by the kit, and the kit’s manual was followed for the library preparation. RNAs were fragmented enzymatically; then, the first and second cDNA strands were generated. The sample was cleaned using AMPure XP beads; then, it was adenylated using the adenylation mix from the kit. Molecular index adapters were ligated to the cDNA; then, it was washed with XP Beads . Finally, the sample was amplified with PCR and washed with XP beads. The Illumina MiSeq reagent kit v3 (150-cycle format) was used for the sequencing; 12 pM from the library was loaded onto a flow cell.The viral transcriptome was also sequenced by using the Illumina SRS approach. The NEXTflexThe amount of the purified RNA samples was measured using a Qubit 4 fluorometer (Thermo Fisher Scientific) and Qubit RNA (broad-range (BR), quantitation of total RNAs) or Qubit RNA HS assay kit (polyA (+) and rRNA-depleted RNAs). Agilent TapeStation 4150 was used to determine the quality of the RNA samples. The amount of the sequencing ready libraries and the quality assessment of the Illumina library was evaluated using the Qubit 4 with Qubit dsDNA high-sensitivity (HS) and the TapeStation, respectively.Base-calling of the raw data from MinION sequencing was carried out with the Guppy software v3.4.5 (ONT). The minimap2 software  was usedhttps://github.com/zsolt-balazs/LoRTIA, accessed on 20 August 2019), using the following steps: (a) Elimination of sequencing reads resulting from partial RT or PCR, as well as artifacts generated by false priming. Non-trimmed read ends were searched for sequencing adapters for the transcriptional start sites (TSS) or the presence of a homopolymer A sequence for the transcriptional end sites (TESs). The first nucleotide not aligning with the adapter was designated as possible TSS, whereas the last nucleotide not aligning with the homopolymer A was designated as putative TES. Any other read start and end positions were discarded. (b) Putative TSSs and TESs were tested against the Poisson distribution to eliminate random start and end positions caused by RNA degradation. The significance is corrected with the Bonferroni method. Features failing to pass qualifying as local maxima or being present in less than 2 reads or in less than 1‰ of the coverage were eliminated from further analysis. The LoRTIA toolkit accepts SAM or BAM files as input and produces GFF files and other processed files as outputs.For transcript annotations, mapped reads were analyzed using the LoRTIA software suite v.0.9.9 (For dRNA sequencing and dcDNA sequencing reads: −5 TGCCATTAGGCCGGG --five_score 16 -- check_in_soft 15 −3 AAAAAAAAAAAAAAA --three_score 16 s Poisson–f true.For o(dT)-primed cDNA reads: −5 GCTGATATTGCTGGG -- five_score 16 --check_in_soft 15 −3 AAAAAAAAAAAAAAA --three_score 16 s Poisson–f true.The LoRTIA suit was set as follows:Bam files were visualized using Geneious and IGV tools. The TSSs obtained by Cage-Seq and TESs obtained by p(A)-Seq  was mappThe raw Illumina reads were trimmed with the Cutadapt tool . The aboThe genome size of the various ASFV strains can significantly vary . We usedIn this work, we report the application of the combined use of SRS (Illumina MiSeq) and LRS (ONT MinION) techniques for the analysis of the ASFV transcriptome . We usedThe average read-length filtered by the LoRTIA software suite was 900.45 bp  . Three aNovel intergenic transcripts with small ORFs. We detected three LoRTIA and four non-LoRTIA small RNA molecules containing short (9–192 bp) ORFs encoded in various intergenic regions  located in an upstream position relative to the canonical ORFs . Our stuNon-coding RNAs (ncRNAs) are specified by RNA genes that are located either at intergenic regions or within protein-coding genes, and they can be encoded by both the positive and negative DNA strands of protein-coding genes. We detected 3 short ncRNAs (sncRNAs) and 42 long ncRNAs (lncRNAs > 200 bp in length), including inter- and intragenic transcripts, antisense RNA (asRNAs), and replication origin-associated RNAs (raRNAs) .intergenic genomic regions   .3′-truncated transcripts are controlled by the same promoters as the overlapping mRNAs, but these RNA molecules are terminated at sequences preceding the canonical stop codons; therefore, they either do not contain ORFs or code very short ones (The ort ones . In ASFVReplication origin-associated RNAs overlap or are located in the vicinity of the replication origins (Oris). These transcripts have been identified in every examined virus [ed virus ; howevered virus . In ASFVAntisense RNAs can, in principle, be controlled by distinct promoters, or they can be generated by transcriptional read-through from adjacent or distal convergent genes or by the overlap of divergently oriented genes. In the latter two cases, antisense sequences only make up a part of an mRNA. Our LRS approach detected seven asRNA fragments with identified TESs, but unannotated TSSs (ted TSSs d. HoweveTSS isoforms (variants) contain the same ORFs but differ in the length of their 5′-UTRs and are usually controlled by distinct promoters. We obtained a comparatively low diversity of TSSs, for which the reason may be the relatively low data coverage and the stringent criteria used for the identification of novel transcripts  signals and 3′-end transcript terminals. In this work, we identified 220 TESs . We identified a total of 22 complex transcripts .We detected 540 parallel, 60 convergent and 19 divergent overlaps . The aveAfrican swine fever virus is one of the most important animal pathogens causing severe losses in animal husbandry. Despite its economic significance, no effective prevention therapy  against this virus is available yet. Understanding the architecture of viral transcriptome is not solely an academic issue because drug or vaccine development is essentially dependent on our knowledge of the fundamental molecular mechanisms.Here we report the first long-read sequencing study on the ASFV transcriptome, which also applies a short-read sequencing approach.The RT and the PCR may generate technical artifacts, such as nonspecific binding of oligo(dT) or PCR primers and template switching. In addition to poly(A) tails, oligo(dT) primers may also hybridize to A-rich regions of RNA molecules and thereby produce spurious transcripts. Additionally, it has been shown that cDNA library preparation of ONT protocol induces read truncation for transcripts containing internal runs of T’s . HoweverThe differences between the amounts of transcripts  at the sOur investigations revealed that the organization of the ASFV transcriptome is more similar to those of the poxviruses than it was expected . AdditioWe identified a number of uORFs  and founIt has recently been shown in several viruses that transcripts embedded into a larger host gene and containing 5′-truncated in-frame ORFs within larger canonical ORFs –, of which they share stop codons—are much more common than it has previously been thought ,16,54. TWe also detected various classes of non-coding transcripts, including intra- and intergenic transcripts and antisense RNAs. The asRNAs are transcriptional read-through products of convergent genes or are generated by divergent overlaps, or they can have their own promoters as in Transcripts can form co-oriented, convergent and divergent overlaps. Polycistronic ASFV transcripts represent a parallel (co-oriented) overlap of RNA molecules encoded by tandem genes. The distinctive feature of ASFV convergent overlaps compared to herpesviruses is the large number of “hard” overlaps  and the large proportion of overlaps in the “soft” overlaps  . AnotherBesides the confirmation of already annotated transcripts , our stuIn sum, our work provides a near-complete assembly of the ASFV transcriptomic atlas. Understanding the genetic regulation provides new insights into the virus pathogenicity. Our results in ASF research imply that targeting the expression of raRNAs may provide an effective antiviral strategy. Another importance of our results is the discovery that the putative nested genes represent an unexpectedly large fraction of coding sequences, which may also be the case in other viruses. Furthermore, this work highlights the necessity for the use of a multiplatform approach in transcriptomic studies.


The dopamine transporter (DAT) serves a critical role in controlling dopamine (DA)-mediated neurotransmission by regulating the clearance of DA from the synapse and extrasynaptic regions and thereby modulating DA action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DATs to alter their actions resulting in an enhancement in extracellular DA concentrations. We previously identified a novel allosteric site in the DAT and the related human serotonin transporter that lies outside the central orthosteric substrate- and cocaine-binding pocket. Here, we demonstrate that the dopaminergic psychostimulant sydnocarb is a ligand of this novel allosteric site. We identified the molecular determinants of the interaction between sydnocarb and DAT at the allosteric site using molecular dynamics simulations. Biochemical-substituted cysteine scanning accessibility experiments have supported the computational predictions by demonstrating the occurrence of specific interactions between sydnocarb and amino acids within the allosteric site. Functional dopamine uptake studies have further shown that sydnocarb is a noncompetitive inhibitor of DAT in accord with the involvement of a site different from the orthosteric site in binding this psychostimulant. Finally, DA uptake studies also demonstrate that sydnocarb affects the interaction of DAT with both cocaine and amphetamine. In summary, these studies further strengthen the prospect that allosteric modulation of DAT activity could have therapeutic potential. The dopamine transporter is a membrane-bound protein present in the presynaptic terminals of dopaminergic neurons in the central nervous system. It belongs to the solute carrier 6 (SLC6) family of transporters and controls the signal amplitude and duration of dopaminergic neurotransmission by transporting extracellular (EC) dopamine (DA) from the synapse back into the presynaptic neuronal terminals . Hence, In an effort to develop therapeutics for substance use disorders, a plethora of DAT ligands have been explored to date that can competitively or noncompetitively block the interaction of cocaine and other DAT-interacting psychostimulants. However, the successful application of such compounds have largely failed due, in part, to their own abuse liability or stimulatory effects and their off-target effects that have impeded their clinical development . DAT ligAquifex aeolicus) leucine transporter (LeuT) -dopamine (32.6 Ci/mmol) and [3H]-serotonin (23.9 Ci/mmol), were purchased from PerkinElmer . Cell culture media and supplements, including penicillin/streptomycin, Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM) with glucose, and scintillation fluid, were obtained from Thermo Fisher Scientific . Transfection reagents TransIT-LT1 and LipoJet reagent were from Mirus Bio LLC  and SignaGen Laboratories , respectively. Reagents for uptake assays and non-radiolabeled substrates were purchased from Sigma-Aldrich . MTSEA-biotin was purchased from Biotium, Inc. . Reagents for uptake assays and non-radiolabeled substrates were purchased from Sigma-Aldrich .Sydnocarb was a gift from Teva Pharmaceuticals . Radiolabeled substrates, -DA) and increasing concentrations of unlabeled substrate (0.05–100-μM DA) were added, and the uptake was allowed to continue for 10 min at room temperature. The uptake was terminated by washing twice with PBS-CM. The cells were solubilized in scintillation cocktail and counted on a microplate scintillation and luminescence counter . Background was obtained from non-transfected cells and subtracted. Data were fitted to a Michaelis−Menten equation using nonlinear regression to obtain Km and Vmax. Transport assays were performed as described previously . Briefly3H]-dopamine to a final concentration of 25 nM. The uptake was allowed to continue for 10 min at room temperature and was terminated by washing twice with PBS-CM. The cells were solubilized in a scintillation cocktail and counted on a microplate scintillation and luminescence counter . Data were fitted to a Hill equation by a nonlinear regression analysis to obtain the IC50 values. Transiently transfected COS-7 cells  were plated in 96-well plates. Uptake experiments were performed 2 days later. The media was removed, and the cells were washed with PBS-CM. Following washing, the cells were incubated for 10 min with various concentrations of sydnocarb, and the uptake was initiated by adding -dopamine transport compared to the vehicle, whereas the difference in the KM values was not statistically significant  and inward-facing open (IFo) states using AutoDock [o conformer, the most favorable binding sites for sydnocarb were exclusively within the EC vestibule. In contrast, no high-affinity binding site was observed in the EC vestibule in the case of the IFo conformer. Therefore, we concluded that the exogenous drug sydnocarb would predominantly bind to the EC vestibule of OFo DAT. We have previously reported similar results with KM822, where it was also shown that KM822 stabilizes the OFo DAT conformation and does not bind to the IFo conformation of hDAT [To assess the binding propensity of sydnocarb, we first performed docking simulations onto hDAT in the outward-facing open  within 60 ± 30 ns to allosterically drive the transition of the transporter to the OF closed (OFc) state, followed by a state occluded to both the EC and IC environments [o hDAT conformation using the GOLD docking software, while the docking of KM822 onto the IFo hDAT conformation resulted in sterically unfavorable interactions [S)-citalopram bound to hSERT (PDB: 5I73) [S)-citalopram, in the simulations and biochemical experiments [Our previous MD simulations of wild-type hDAT, starting from the OFronments ,45. In cractions . Both thB: 5I73)  and agreeriments . Our MD simulations from KM822-Run1 showed that KM822 folds within the A2 site with the triazinoindole ring system pointing towards the EC side, making hydrophobic interactions with F217, I469, and Y548 . The aryWhen comparing the sydnocarb-binding pose within A2 C with thOverall, this detailed examination showed that the allosteric sites A2 and Run1-site were distinct from the substrate-binding site S1  method . We prevo conformation of hDAT, mimicking the docked KM822-hDAT conformation. In order to provide experimental evidence for these observations, we used the mutants Y156F and Y335A to determine the preferred DAT conformation for various DAT-interacting compounds [o hDAT conformation and that they do not bind to the inward-facing conformer. Our docking experiments using AutoDock suggested that sydnocarb prefers to bind to the OFompounds ,48,49. Mompounds . These eNext, we examined the effects of sydnocarb on the dose-dependent cocaine and amphetamine inhibition of hDAT-mediated DA transport in DAT transfected COS-7 cells. We observed that the presence of sydnocarb dose-dependently decreased the potency of cocaine in the dose-response DA transport inhibition assays . The DATo conformation of hDAT. Sydnocarb also reduces the potency of hDAT to interact with the highly addictive psychostimulants cocaine and amphetamine in cell-based assays. Further works investigating the structure–activity relationship of sydnocarb will provide a clearer picture of the potential importance of developing sydnocarb as a tool for understanding the allosteric modulation of hDAT function. Better options and new insights for treating psychostimulant use disorders are imperative, as the current treatments are generally known to be ineffective. The results presented in this paper provided strong empirical evidence that sydnocarb is an allosteric modulator of hDAT and that it could serve as a promising lead molecule for developing novel therapeutics against addiction. This study further supported the therapeutic relevance of engaging potential allosteric sites within the EC vestibular region of hDAT using small molecules. We used docking and MD simulations and biochemical methods to understand the molecular basis of the pharmacological potential of sydnocarb as an hDAT modulator and compared the results with that of KM822. Our previous studies showed that KM822, one of the molecules that binds to the allosteric domain, modulated the interaction of cocaine with hDAT in various in vitro and in vivo models of addiction. The mechanistic studies herein suggested that sydnocarb, like KM822, binds to the EC allosteric domain away from the orthosteric site of hDAT and closer to the EC salt bridge R85-D476, stabilizing the OF


Nevoid hyperkeratosis of nipple and areola (NHNA) has been classified into 3 types: type 1 is associated with an epidermal nevus; type 2 with various dermatoses such as the Darier disease (DD), chronic eczema, and cutaneous T-cell lymphoma; type 3 is of unknown etiology. Despite the multifactorial association, a consistent morphological feature of NHNA is the presence of brown verrucous plaques involving the nipple and areola area, making the clinical distinction difficult [A 25-year-old male with skin type V presented with multiple variably pigmented lesions all over the body since the age of 5. He reported a history of swelling affecting the bilateral nipple-areola complex (NAC) for 1 year. Lesions were foul-smelling and exacerbating during summer. He did not report a similar family history. On cutaneous examination, the bilateral NAC showed erythematous to gray verrucous plaques that hindered the visibility of the nipples . DermoscDermoscopic features of NHNA are sparsely reported. Mazzella et al. reported multiple blue-gray globules and leaf-like areas in the case of type 3 NHNA . In the Here we reported a characteristic dermoscopic pattern found in a case of DD associated NHNA, which can be valuable for future diagnosis and for differentiating it from other NHNA causes.


Importantly, DSF/Cu inhibits several molecular targets related to drug resistance, stemness, angiogenesis and metastasis and is thus considered as a novel strategy for overcoming tumour recurrence and relapse in patients. Despite its excellent anticancer efficacy, DSF has proven unsuccessful in several cancer clinical trials. This is likely due to the poor stability, rapid metabolism and/or short plasma half-life of the currently used oral version of DSF and the inability to form Cu(DDC)2 at relevant concentrations in tumour tissues. Here, we summarize the scientific rationale, molecular targets, and mechanisms of action of DSF/Cu in cancer cells and the outcomes of oral DSF ± Cu in cancer clinical trials. We will focus on the novel insights on harnessing the immune system and hypoxic microenvironment using DSF/Cu complex and discuss the emerging delivery strategies that can overcome the shortcomings of DSF-based anticancer therapies and provide opportunities for translation of DSF/Cu or its Cu(DDC)2 complex into cancer therapeutics.Copper (Cu) plays a pivotal role in cancer progression by acting as a co-factor that regulates the activity of many enzymes and structural proteins in cancer cells. Therefore, Cu-based complexes have been investigated as novel anticancer metallodrugs and are considered as a complementary strategy for currently used platinum agents with undesirable general toxicity. Due to the high failure rate and increased cost of new drugs, there is a global drive towards the repositioning of known drugs for cancer treatment in recent years. Disulfiram (DSF) is a first-line antialcoholism drug used in clinics for more than 65 yr. In combination with Cu, it has shown great potential as an anticancer drug by targeting a wide range of cancers. The reaction between DSF and Cu ions forms a copper diethyldithiocarbamate complex (Cu(DDC) Cancer is a prominent cause of death worldwide which places an increasing burden on health and socioeconomic systems. According to the estimates provided by World Health Organization (WHO) in 2019, cancer is already the first or second cause of death before the age of 70 in 112 of 183 countries and its prevalence is expected to increase . It is pIn addition to surgery and radiation, chemotherapy which uses broad-spectrum cytotoxic drugs that do not differentiate cancer cells from normal cells, remains as the main arm of cancer treatment, despite its toxicity and significant side effects . The disBy the turn of the millennium, insights into the genomic landscapes of cancers led to the development of drugs that selectively target cancer cells while sparing normal cells, hence having high potency and fewer adverse effects . Over thOwing to the clinical success of platinum drugs in a wide range of cancers and the requirement for reduction of treatment costs, there has been renewed interest in the development of novel metallodrugs in the field of inorganic drug development . While tConsidering the reduction of development costs and affordability as a priority, repurposing old metal binding drugs to form complexes with Cu to selectively target cancer cells has been rigorously investigated in the past years . One sucHere, we review the roles for Cu in cancer progression and the potential of harnessing copper homeostasis pathways in cancer cells with the Cu-binding activity of DSF and its derivatives, with a focus on recent developments in the translation of DSF for cancer therapy.Dietary supplementation and absorption in the stomach, duodenum and upper small intestine is the main source of Cu in humans. Most of the absorbed Cu is stored in the liver and the Cu concentration in serum is approximately 12.0–25.0 μmol/L and varies in tissues between 15.0 and 180.0 μmol/g . In physMany studies have indicated that the levels of Cu in both serum and tumour tissues are remarkably elevated in cancer patients in comparison to healthy individuals as summarised in Although the underlying mechanisms alter the Cu levels in the serum of cancer patients remain unclear, the role played by Cu in etiology and progression of tumours has been widely studied in the past two decades. Exposure to high levels of Cu has been linked with pancreatic neuroendocrine tumours and prostate cancer . As a meRecently it has been shown that enhanced levels of Cu upregulate the expression of programmed death ligand 1 (PD-L1) in tumour cells and modulate the signalling pathways that mediates the PDL-driven immune escape by cancer cells . It has Cu has the ability to switch on many pro-angiogenic responses and aid the proliferation and mobility of endothelial cells, which initiates angiogenesis that provides oxygen and essential nutrients to tumours . Cu is cin vitro and in vivo as well as in clinical trials .in vivo, that might explain the potential intolerance to alcohol for a longer period of time 2 enables its systemic absorption to take place along the upper GI tract (Both DSF and its metabolite diethyldithiocarbamate (DDC) are stable in basic environments, but unstable in acidic conditions. Upon ingestion, more than 99% of DSF is quickly converted to its corresponding thiol metabolite DDC in the acidic environment of the stomach. Both forms are rapidly absorbed through the gastrointestinal mucosa into the portal circulation and enriched in liver where both DSF and DDC are rapidly metabolised and degraded. DDC, due to its hydrophilic polar nature, is easily reduced to diethylamine (DEA) and carbon disulphide (CS2). CS2 is reactive with endogenous nucleophilic groups including thiols, amino acids and proteins and can be oxidized to form carbonyl sulphide (COS). COS can be further oxidised to form carbon dioxide and sulphur radicals, resulting in a permanent inhibition of microsomal mono-oxygenases. Further, CS2 can be involved in re-synthesis of DDC by reacting with DEA  of time . Both DS of time . The metGI tract . The intDSF, after being absorbed into circulation is immediately broken into DDC monomers by the glutathione reductase system in red blood cells. Free DDC quickly binds to thiols of proteins, specifically to albumins, to form disulfides. DDC is also a substrate for Phase II metabolism that is mediated by an S-methyl-transferase, resulting in the formation of S-methyl diethyldithiocarbamate (S-Me-DDC) and S-glucuronide of DDC. Oxidative biotransformation of S-Me-DDC forms S-diethylthiomethylcarbamate (S-Me-DTC), which could be further oxidized to S-Me-DTC-sulphoxide and S-Me-DTC-sulphone metabolites, respectively . All theEthanol metabolism in the liver predominantly relies on the action of ALDH isozymes and a minor amount of ethanol is metabolised through P450 mono-oxygenases and catalases. Upon intake of alcohol, ethanol oxidation produces acetaldehyde which is further oxidised by ALDH to form acetate. Two important ALDH isoenzymes involved in this process are ALDH1 and ALDH2 . SulphoxIn addition to ALDH, DSF is also known to inhibit other enzymes involved in vital metabolic pathways such as glycolysis, tricarboxylic acid cycle, pentose phosphate shunt, glutathione system, and catecholamine synthesis through S-S linkage or by chelation of copper or zinc from the active sites of the enzymes . DSF canin vitro conditions, a mixture of DSF and Cu immediately results in a highly oxidized intermediate form of DDC termed as bis-tetrathiolane dication (Bitt-42+), followed by subsequent spontaneous decomposition of small amount of DSF to its anionic chelate form DDC, which on further redox reaction with Cu2+ forms a stable complex Cu(DDC)2. This redox reaction and the Fenton chemistry involved in the formation of Cu(DDC)2 complex results in the generation of ROS, which induces apoptosis in cancer cells. DSF/Cu, has been shown to be cytotoxic to cancer cells and has the ability to eradicate cancer stem cell (CSC) populations in various cancers with little or no toxicity to normal cells (The anticancer activity of DSF has been demonstrated in various cancer cells models and is shown to be strongly dependent on formation of Cu(DDC)2 complex with divalent metal ions such as Cu. In al cells . DSF/Cu al cells . There aal cells . It can al cells .2 get accumulated in the cancer cells and induce ROS generation leading to apoptosis in cancer cells . These ClC-3 channels are highly functional and over-expressed in cancer cells as compared to normal cells, and DSF/Cu complex is only able to trigger activation of over-expressed ClC-3 channels. Therefore, DSF/Cu complex specifically induce apoptosis in the cancer cells, through targeting over-expressed ClC-3 channels, without affecting the normal cells  trigger . Apoptos trigger . It was  trigger . Chiba e pathway . Nasopha pathway . Zhang e pathway . DSF/Cu al cells . In summA functional proteasome system maintains the synchronized expression of cell cycle-regulatory and apoptosis-control proteins. Hence the proteasome-mediated protein degradation is an important cellular homeostatic system, and in general cancer cells are more sensitive to alterations in this proteasome inhibition than normal cells. Bortezomib was the first clinically approved 20S proteasome inhibitor drug used for its antitumor activity against several types of cancers . ProteasThe protein complex, nuclear factor-kappa B (NF-κB), has a close relationship to cancer cells by acting as a key regulator of multiple pathways necessary for survival, stemness, resistance, angiogenesis and metastasis . NF-κB iHowever, a recent study argued that the DSF/Cu complex (Cu(DDC)2 or CuET) is not a direct inhibitor of proteasomes, instead CuET targeted the p97-NPL4-UFDI pathway upstream to proteasome. It was shown that CuET binds to the NPL4, resulting in aggregation of NPL4 and deactivation of P97 segregase which leads to accumulation of misfolded proteins in the cells and ultimately cell death . It was via NPL4 targeting. The main target of CuET in p97 segregase pathway is NPL4, a key component of p97 segregase. The interaction of NPL4 and CuET results in the formation of protein aggregates and immobilization of otherwise very mobile NPL4-p97 complex which triggers the cellular heat shock response and endoplasmic reticulum (ER) stress/unfolded protein response, eventually leading to cell death . CuET caThe same group also discovered that cells lacking BRCA1 and BRCA2 were particularly sensitive to DSF/Cu or CuET treatment. CuET induced replication stress-associated DNA damage in several cancer cell lines and increased γH2AX, as an indicator for DNA double-strand breaks in these cells (DSB). Homologous recombination (HR), a DSB repair mechanism, requires BRCA1 and BRCA2 for its activity. Interestingly, a co-localization of ATR with immobilized NLP4 aggregates was found after CuET treatment. Moreover, CuET interfered with the activation of the RPA-ATRIP-ATR-CHK1 repair pathway, by suppressing the ATR kinase despite of high replication stress, induced by ssDNA aggregates .Cancer stem cells (CSC) are highly resistant to several conventional chemotherapies and therefore, these cells are responsible for the cancer recurrence . ALDH bein vitro toxicity in cancer cells, there was almost no positive clinical data published in cancer patients https://clinicaltrials.gov/ct2/results?term=disulfiram+AND+cancer&Search=Search). A summary of all the clinical trials using oral DSF is given in Although DSF shows high 1) The instant ROS mediate damages induced by the reaction between DSF and Cu; 2) A delayed killing induced by the end product, Cu-DDC  particles to encapsulate the drug of interest . SeveralPolymeric nanoparticles are simple in design and encompass extensive structural diversity, outstanding bio-compatibility and stability . One of in vitro and in vivo breast cancer mouse models  in vitro . DSF hasin vitro . The Au-2 and Zn(DDC)2 are hydrophobic; nanoparticle albumin bound technology may be the key to develop an injectable, long circulating formulation.Albumin is a promising macromolecular drug carrier as it is biocompatible, biodegradable, non-immunogenic and soluble in water to allow for injection. In addition to this, it is possible to modify the surface of albumin nanoparticles for targeting. Previously, nanoparticle albumin bound technology has been used to solubilize the hydrophobic cancer drug paclitaxel (Abraxane) which is now a first line drug for PDAC . Since the complexes Cu(DDC)2 and Regorafenib (Rego) were successfully encapsulated in bovine serum albumin (BSA) nanoparticles via denaturation of BSA by urea . The formulation targeted secreted protein acidic and rich in cysteine (SPARC) receptors. Furthermore, mannose receptors were targeted by modifying the surface of the BSA nanoparticles. It had enhanced anti-cancer efficacy in an animal model bearing resistant tumours via apoptosis, upregulation of intracellular ROS, anti-angiogenesis and tumor-associated macrophage repolarization. These nanoparticles could also effectively cross the blood brain barrier to deliver Cu(DDC)2 and Rego to treat gliomas in mice  which are cyclic oligosaccharides that can form truncated-cone-shaped macrocyclic molecules that possess hydrophobic cavities. These cavities can solubilize hydrophobic drugs and Suliman et al. (2021) developed a CD formulation of Cu(DDC)2 and demonstrated stability for 28 days with enhanced cytotoxicity against resistant breast cancer cell lines (Cu(DDC) in mice . The optll lines .Together, all the above evidence has indicated that using nanotechnology-based formulation strategies can significantly improve the half-life, stability, bioavailability and it is possible to achieve delivery of DSF at therapeutic doses in cancer patients.The success of platinum drugs has led to the search for metal complex-based anticancer therapeutics. The search for novel metallodrugs or metal complexes that target cancers is still a field of high interest in the pharmaceutical sector. The fundamental role of Cu in aiding cancer progression and the dysregulation of Cu homeostasis in cancers has been well documented in both preclinical and clinical settings. With Cu as an emerging novel target, there has been major advancements in the development of Cu coordination complexes as anti-cancer therapeutics. Use of Cu chelators and Cu ionophores have yielded encouraging anticancer activities in many preclinical models. Particularly many drugs that are used for other indications have been identified and repurposed as Cu coordination drugs, not only to achieve better antitumour efficacy, but also to enhance the efficacy of first line anticancer drugs such as platins.in vitro studies or in vivo pre-clinical animal models. Although many of these delivery strategies demonstrated great potentials for DSF as an anticancer agent, these delivery systems must be extensively evaluated with further studies to gather complete preclinical information such as safety, toxicology, pharmacokinetic and optimal physicochemical properties which are crucial to facilitate clinical translation.Among many repurposed metal chelator drugs, DSF has a great promise and potential to be used as an anticancer agent. With its anti-angiogenic potential, and the ability to target CSCs and synergistically potentiate many first line conventional drugs, DSF could provide new opportunities to develop effective combination therapeutic strategies. DSF also has a great potential be an effective immunomodulatory drug by regulating NF-kB signalling or by reducing PDL-1 expression by Cu chelation. In this review we have summarised the effectiveness of DSF as an anticancer agent, which applies to a wide range of cancers. Despite several clinical trials, no reliable efficacy of DSF as an anticancer drug for tumours has been observed due to the lack of biological availability and short half-life of the currently available oral DSF. Therefore, the development of an appropriate formulation is critical for the successful translation of DSF into cancer indication. Here we summarized different delivery strategies that have been developed for DSF-based cancer therapy. Most of the identified or developed drug delivery systems for DSF are at the proof-of-concept stage either in Although many different delivery strategies using novel materials indicated additional benefits, the use of FDA approved excipients could minimize regulatory hurdles and considerably reduce the preclinical development time. Moreover, the development of encapsulation strategies should be suitable for scale-up production for manufacturing and commercialization. In addition to these technical issues, there are many regulatory and marketing hurdles for commercialization of DSF based anticancer product, predominantly due to the lack of patent protection on DSF.Therefore, pharma companies do not show much interest to invest in clinical trials for DSF-based anticancer therapeutics. The development of novel formulations of DSF together with appropriate strategies for exploitation of regulatory guidelines, would lead not only to the development of intellectual properties and enhanced anticancer efficacy, but also will lead to the successful development and clinical translation of DSF as an anticancer product for commercialization.


This systematic review and meta-analysis aimed to investigate the role of alendronate combined with step 2 of periodontal therapy in reducing probing pocket depth, improving clinical attachment level, and reducing bone defect depth in intra-bony and inter-radicular defects.RCTs with more than 6 months follow-up were included in this study. Risk of bias assessment was performed using the Cochrane collaboration tool. In addition, meta-analysis and trial sequential analysis were used to aggregate the available evidence.Seven studies met the inclusion criteria and were included in the systematic review. Topical application of alendronate during second step of periodontal therapy significantly improved PD and CAL.Local application of alendronate may confer a beneficial effect when applied during step II of periodontal therapy even if long term studies are needed to confirm these results.Considering the emerging role of host-inflammatory response in treatment of periodontitis and the antiresorptive and osteostimulative properties of bisphosphonates, several studies are focusing on the role of alendronate as an addition to non-surgical periodontal therapy.The online version contains supplementary material available at 10.1186/s12903-022-02044-1. Periodontitis is an inflammatory disease affecting tissues surrounding teeth, characterized by destruction of connective tissue attachment and alveolar bone . It is mA systematic review protocol was written in the planning stages and both the Cochrane Handbook and the PRISMA  statement were followed for the planning and reporting of the review. In addition, the protocol of this systematic review was registered on the PROSPERO database (registration code: CRD42021223883).This review was performed aiming to answer the following PICO question: “Do intra-bony and inter-radicular defects (Participants) heal better with the adjunct of 1% alendronate (Intervention) to SRP instead of placebo (Comparison) in terms of PD, CAL and bone defect depth (Outcomes)?Type of studies: Randomized controlled trials with a follow-up of at least 6 months. A shorter follow up was not considered as it would be unlike to reflect a meaningful difference in treatment response between test and control; (2) Types of participants: Adults (> = 18 years old), systematically healthy individuals diagnosed with periodontitis; (3) Types of intervention: Studies evaluating the adjunctive use of alendronate 1% gel administered locally during step 2 of periodontal therapy in intra-bony and inter-radicular defects; Comparison: patients receiving placebo or none adjunct treatment during step II of periodontal therapy; (4) Outcomes: Primary outcome: reduction in PD, CAL gain and bone defect depth. Hence, the following exclusion criteria were considered: (1) Non RCT or RCTs with a follow up < 6 months were excluded; (2) Studies involving less than 20 sites per group; (3) studies including participants with systemic diseases or that were taking medications were excluded.Only studies fulfilling the following inclusion criteria were considered eligible for inclusion in this review: (1) Studies were identified through an online search on PubMed, Scopus, and Web of Science. The search strategy included terms related to the population and the intervention. A combination of MESH terms and Free Text words combined with Boolean operators; for example in PubMed the following string was used:  AND (‘osseous defects’ OR ‘intrabony defects’ OR ‘infrabony defects’ OR ‘furcation defects’ OR ‘furcation’ OR ‘periodontitis’))). A manual search was performed through several scientific journals, namely: Journal of Dental Research, Journal of Clinical Periodontology, Journal of Periodontology, International Journal of Periodontics and Restorative Dentistry, Journal of Oral and Maxillofacial Surgery. The bibliographies of pertinent review articles and studies finally included for data extraction were also screened.Eligibility of studies was assessed by two independent authors in a standardized manner . In the first round, records were screened by only reading title and abstract of publications. The studies assessed as eligible were included in the second round and underwent full-text reading. Only studies fulfilling the inclusion criteria were considered eligible and included in the review for the subsequent data extraction. Disagreements between authors were solved through discussion and a third author (GT) evaluated the agreement between reviewers by calculating a value of K-statistic. In addition, data extraction and collection were performed by two authors  in a joint session using an ad hoc extraction sheet.The Cochrane Collaboration Tool was used for risk of bias assessment in the included studies . The anaI2). Data were pooled with a fixed-or a random-effect model on the basis of an I2 lower or upper the cut-off of 50%. The inverse of variance test was used to analyze the overall effects. We combined split-mouth and parallel designs as suggested by Elbourne et al. [For the pooled analysis of PD reduction, CAL gain and bone defect depth reduction, the mean difference (MD) and its standard error (SE) between the two groups were calculated. The presence/absence of heterogeneity was assessed by means of the Higgins Index  when alendronate was topically applied during step II periodontal therapy compared to placebo; MD = 2.01 . Such results were characterized by a high rate of heterogeneity (I2 = 82%), for such reason a random effects model was used for data interpolation. A subgroup analysis was performed based on study design  to test for a possible influence of study design on the analyzed outcome. The effect estimate for parallel group studies was 2.11  and for split-mouth 0.30  and although both groups showed a benefit for alendronate compared to placebo an important difference was detected. Results of the TSA confirmed the previously performed meta-analysis with the z-curve crossing the lower alpha spending boundary, in addition the meta-analysis was characterized by a good power of evidence since the RIS (74 patients) was crossed . Such rp < 0.00001) when alendronate was topically applied, MD = 1.72 . Such results were characterized by a high rate of heterogeneity among studies (I2 = 88%) and for this reason a random effect model was used for data interpolation. A sub-group analysis was performed based on study design ; the effect estimate for parallel group studies was 1.82  and for split-mouth 0.90 , also in this case study design influenced the effect size of results. The TSA confirmed the previously performed meta-analysis with the z-curve crossing the lower alpha spending boundary, in addition the meta-analysis was characterized by a good power of evidence since the RIS (74 patients) was also crossed . TSA confirmed such findings with the z-curve crossing the lower alpha spending boundary and RIS threshold (99 patients). No differences were detected between intra-bony and inter-radicular defects (p = 0.18) , with results characterized by a high rate of heterogeneity (Evidence by subgroups was qualified using the GRADE approach. Low quality of evidence supports the beneficial effect of alendronate 1% as an adjunct to second step of periodontal therapy in the treatment of intra-bony and inter-radicular defects. The level of evidence was downgraded due to inconsistency results of the included studies. Results of the GRADE evaluation are summarized in Additional file This systematic review aimed to provide a clear focus on the role of alendronate 1% as an adjunct to second step of periodontal therapy in treatment of intra-bony and inter-radicular defects. Since the introduction of host-modulators in the treatment of periodontal disease , severalResults of the present study showed that the topical application of Alendronate gel 1% can provide a significant benefit in PD reduction, CAL gain and bone defect depth reduction when applied after non-surgical periodontal treatment. A significant difference between parallel groups and split-mouth design was detected when subgroup analysis was performed for PD reduction and CAL gain. No significant differences were detected between intra-bony and inter-radicular defects when subgroup analysis was performed for bone defect depth reduction. BPs are often administered orally for the treatment of post-menopausal osteoporosis showing a wide range of adverse events including gastro-intestinal side effects, acute phase syndrome, secondary hyperparathyroidism, hypocalcemia, musculoskeletal pain and osteonecrosis of the jaw; therefore local application of alendronate might appear to be safer and devoid of side effects since none of the studies had drop-out patients that seemed to be related to local treatment; furthermore local administration offers the advantage of reaching a higher concentration of drug at the osseous defect with a reduced dosage . HoweverFirst of all, only two of the included studies , 20 repoOf the seven included studies, only one provided more detailed information about the subgingival instrumentations performed ; in the We did not set the smoking status as an exclusion criterion, so this factor should be considered in the outcome of treatment, even if the use of alendronate as a host modulator in intraosseous defects could be particularly important for smokers whose healing capacity is impaired , 34. SinBased on the results available from these RCTs, this review shows that local delivery of alendronate seems to be effective in improving PD, CAL and bone defect depth. However, studies conducted by different research groups and on a geographically more heterogeneous population with a more standardized protocol are necessary to confirm these findings. Therefore, even if topical administration of Alendronate could have a potential beneficial effect on periodontitis clinical parameters, multicentric studies with a longer follow-up are needed to clarify this point.Additional file 1. Results of the GRADE evaluation.


Researchers have increasingly paid attention to the neural dynamics of depression. This study examined whether self-dependent neural variability predicts recovery from depressive symptoms. Sixty adults with depressive symptoms who were not officially diagnosed with major depressive disorder participated in this study. Participants completed functional magnetic resonance imaging (fMRI) scanning, including a resting-state and a self-reflection task. The fMRI data were used to estimate neural variability, which refers to the temporal variability in regional functional connectivity patterns. Participants then completed the Self-Construal Scale and the Beck Depression Inventory (BDI). The change in BDI scores over 3 months indicated the degree of recovery from depressive symptoms. Self-construal moderated the effects of general neural variability on predicting recovery from depressive symptoms. Interdependent individuals became less depressive with higher general neural variability, but the relationship was not significant in independent individuals. The differences in neural variability between self-related and other-related conditions also predicted recovery from depressive symptoms. The regions contributing to the prediction were mainly distributed in the default-mode network. Based on these results, the harmony between individuals’ neural dynamics and self-concept is important for recovery from depressive symptoms, which might be a foundation for individualized treatment and counseling. Depression has affected over 264 million people worldwide . Based ovs non-self-judgment elicits neural activity in the dorsal medial frontal gyrus (MFG) and the dorsolateral prefrontal cortex (DLPFC) and enhances functional connectivity between them in patients with MDD , are closely related to depression  was used as the control condition. Six scans with seven blocks in each scan were performed. Each block presented a type of judgment. The blocks were presented in a random order and with a 10-s interval. Each item was presented for 2 s and followed by a 1-s fixation.During fMRI scanning, the participants completed a 5-min resting-state and a self-reflection task . During After scanning, participants completed the Self-Construal Scale . The Sel3. During resting-state scanning, the participants were asked to keep their eyes open.We used a GE Signa MR750 3.0T scanner with a standard head coil to acquire the fMRI data. The data were acquired using T2-weighted, gradient-echo, echo-planar imaging (EPI) sequences with the following parameters: repetition time (TR) = 2000 ms, echo time (TE) = 30 ms, flip angle (FA) = 90°, field of view (FOV) = 240 × 240 mm, matrix = 64 × 64 × 32 and spatial resolution = 3.75 × 3.75 × 5 mmvs no removal) to test the robustness of the prediction effects across nuisance covariate removal strategies (see supplementary results).A standard preprocessing procedure was performed on the fMRI data using the Data Processing Assistant for Resting-State fMRI (DPARSF) toolbox . The datA 264-node atlas  of the matrix represented the functional connectivity between a specific node and the remaining nodes across the whole brain. For each node, we compared the functional connectivity matrices across different windows of time using Pearson’s correlation analysis. General neural variability of the node was calculated by subtracting the mean of correlation coefficients from one. The following formula was used:We used the fMRI data collected during the self-reflection task to estimate the difference in neural variability between the self-related conditions and other-related conditions. The neural variability of each condition was calculated by comparing regional functional connectivity matrices across different scans under the same condition. The procedure was similar to the estimation of general neural variability. The difference in neural variability was calculated by subtracting the mean neural variability in the three other-related conditions from the mean neural variability in the three self-related conditions.We used the leave-one-out cross-validation method to study whether self-dependent neural variability predicted recovery from depressive symptoms in a novel individual . In eachBefore the prediction analysis, the dot product of normalized general neural variability and normalized interdependence was defined as the interaction between general neural variability and interdependence. We used the interaction between general neural variability and interdependence to study whether self-construal moderated the effects of general neural variability on predicting the recovery from depressive symptoms in a novel individual.In the feature-selection stage, based on the training set, we performed Pearson’s correlation analysis between the interactions in each node and recovery scores (feature-selection threshold = 0.05). The nodes whose interactions were significantly positively correlated with recovery were selected as positive features, and the significantly negatively correlated nodes were negative features. In addition, we used different feature-selection thresholds (thresholds = 0.05 and 0.01) to test the robustness of the prediction effects across feature-selection thresholds (see supplementary methods and results).In the model establishment stage, positive features or the opposite number of negative features were averaged, resulting in the positive feature interaction strength or the negative feature interaction strength, respectively, and they were averaged together, resulting in the total feature interaction strength. Simple linear regression analyses were conducted to construct the relationships between the three feature interaction strengths and recovery, resulting in three models: the total model, positive model and negative model.In the prediction stage, the same features as the training set were extracted from test set, and the three interaction strengths were substituted in the corresponding model, resulting in the predicted recovery for the participant in each of the three models.In the model evaluation stage, the correlation coefficient between the predicted recovery calculated using the model and the observed recovery assessed using the scale was defined as the predictive power of the model. Only significant positive predictive power indicated that the prediction was successful. The nodes that were selected as features in more than 95% of the iterations were regarded as important nodes. We performed permutation tests to further confirm the significance of the predictions (see supplementary methods).The procedure was similar to the prediction analyses of the interaction between general neural variability and interdependence except that the interaction was replaced with the differences in neural variability between self-related and other-related conditions. Moreover, for the domain-specific prediction effect, we used the differential neural variability between self-related conditions and other-related conditions in which participants made judgments about mental attributes, physical attributes or social attributes to perform the prediction analyses (see supplementary methods).r = 0.31, P = 0.016, r = 0.32, P = 0.012). In the negative model, no feature was selected in at least one iteration. The prediction effects were robust across nuisance covariate removal strategies  and feature selection thresholds  (see supplementary results). However, general neural variability itself failed to predict recovery from depressive symptoms in the three models . The results indicated that self-construal moderated the effects of general neural variability on predicting recovery from depressive symptoms in individuals. Permutation tests further confirmed that the observed predictive power significantly differed from the predictive power in the null distribution  and DMN (n = 5).The interaction between general neural variability and interdependence successfully predicted recovery from depressive symptoms in the total model . The simple effects were consistent on the high-depression group  and the low-depression group . Based on the results, interdependent individuals became less depressive if they had higher general neural variability.We conducted a simple effects analysis with the mean general neural variability of the important nodes to examine the relationship between general neural variability and recovery from depressive symptoms in individuals with high interdependence (M + s.d.) and in individuals with low interdependence (M − s.d.). General neural variability was positively correlated with recovery from depressive symptoms . In the positive model, no feature was selected in at least one iteration. The prediction effects were robust across feature selection thresholds . However, the differences in neural variability between the self-related conditions and font condition  or the differences in neural variability between the other-related conditions and font condition  failed to predict recovery from depressive symptoms. Thus, the difference between self-related neural variability and other-related neural variability predicted recovery from depressive symptoms in individuals. Permutation tests further confirmed that the observed predictive power significantly differed from the predictive power in the null distribution .The differences in neural variability between the self-related conditions and other-related conditions during the self-reflection task successfully predicted recovery from depressive symptoms in the total model . The prediction effects were robust across feature selection thresholds . Thirty-four important nodes were identified, all of which were negative features . However, the differences in neural variability in the physical condition or in the social condition failed to predict recovery from depressive symptoms . The results indicated the importance of the mental aspect of the self.Moreover, the difference in neural variability between the self-related conditions and other-related conditions in which participants made judgments about mental attributes marginally significantly predicted recovery from depressive symptoms in the total model . In the mental condition, the prediction effect was significant in the high-depression group .We examined the robustness of the predictive models in the high-depression group and in the low-depression group using the important nodes as features. The differences in neural variability within the important nodes successfully predicted recovery from depressive symptoms in both the high-depression group . Interdependent individuals with higher general neural variability in the resting state recovered better from depression, while independent individuals with higher neural variability showed no recovery. Interdependent individuals interpret their thoughts, feelings and actions in association with connectedness and integration with the external social environments, and independent individuals comprehend their thoughts, feelings and actions concerning separateness and uniqueness from within rather than reference to others . InterdeMoreover, the differences in neural variability between self-related conditions and other-related conditions successfully predicted recovery from depressive symptoms. A greater difference in neural variability between self-related conditions and other-related conditions was related to better recovery from depression symptoms. Analogously, previous research has emphasized the important role of self-related processing in depression, which has previously been reported in several studies  when they made judgments on personality characteristics describing themselves rather than general desirable characteristics  was induced in Chinese participants than in Danish participants when making self-judgments of social attributes (One limitation of our study was that all the participants were Chinese. As previously shown, interdependent self-construal is adaptive and dominant in Eastern Asian culture, but independent self-construal is adaptive and dominant in Western culture . Danish Taken together, our findings suggest that self-dependent neural variability predicts recovery from depressive symptoms in individuals. The effects of general neural variability on predicting recovery from depressive symptoms may be moderated by self-construal. Interdependent individuals with higher general neural variability in the resting state recover better from depressive symptoms. The difference between self-related neural variability and other-related neural variability predicted the recovery from depressive symptoms, especially in the mental domain. Our results provide the inspiration to understand and improve individuals’ mental health, which might be a foundation for individualized treatment and counseling in the future.nsab050_SuppClick here for additional data file.