Influenza virus infection remains a major public health challenge, causing significant morbidity and mortality by annual epidemics and intermittent pandemics. Although current seasonal influenza vaccines provide efficient protection, antigenic changes of the viruses often significantly compromise the protection efficacy of vaccines, rendering most populations vulnerable to the viral infection. Considerable efforts have been made to develop a universal influenza vaccine (UIV) able to confer long-lasting and broad protection. Recent studies have characterized multiple immune correlates required for providing broad protection against influenza viruses, including neutralizing antibodies, non-neutralizing antibodies, antibody effector functions, T cell responses, and mucosal immunity. To induce broadly protective immune responses by vaccination, various strategies using live attenuated influenza vaccines (LAIVs) and novel vaccine platforms are under investigation. Despite superior cross-protection ability, very little attention has been paid to LAIVs for the development of UIV. This review focuses on immune responses induced by LAIVs, with special emphasis placed on the breadth and the potency of individual immune correlates. The promising prospect of LAIVs to serve as an attractive and reliable vaccine platforms for a UIV is also discussed. Several important issues that should be addressed with respect to the use of LAIVs as UIV are also reviewed. Influenza viruses have posed serious threats on human public health worldwide despite development of effective vaccines and antiviral drugs. Each annual influenza epidemic affects 5−15% of the population and causes 3−5 million cases of hospitalization, claiming 290,000−650,000 lives worldwide [Extensive efforts to develop a universal influenza vaccine (UIV) that provides broad protection against diverse influenza viruses have been made worldwide . InductiAmong various types of LAIVs developed so far, cold-adapted LAIVs (CAIVs) are currently licensed for clinical uses in humans. CAIVs are established by serial passages of parental influenza viruses at low temperatures in embryonated chicken eggs or chicken cells, making the virus less replicative at normal and elevated body temperatures. The resulting cold-adapted virus exhibits cold-adapted (ca), temperature-sensitive (ts), and attenuated (att) phenotypes and, thus, can be used as a safe CAIV donor strain. Genetic and phenotypic analysis have revealed that mutations in the polymerase genes and the NP gene are crucial for the expression of the ca, ts, and att phenotypes ,6,7,8. AReverse genetics technology has made it possible to introduce targeted mutations into the genome of influenza viruses for generation of a number of genetically engineered influenza viruses. This resulted in various attenuation strategies for the development of novel types of LAIVs . InfluenIt is well-documented that natural infection with an influenza virus induces broadly reactive T cell responses that provide cross-protection not only against homologous but also against drifted strains and even antigenically distant viruses with different subtypes . The samInduction of broadly neutralizing antibodies (bnAbs) specific to conserved HA stalk region have been considered as the key to the development of a UIV. To date, a number of antibodies targeting the HA stalk capable of neutralizing a wide range of influenza strains and subtypes have been characterized . HA stalSeveral studies have shown that LAIVs also induce HA stalk antibodies in animals and humans. A mouse study showed that prime-boost vaccination with LAIVs each carrying different subtypes of full-length HA proteins induced robust HA stalk antibodies . AlthougThe M2 protein is the third most abundant surface protein of an influenza virus. M2 is also expressed on the surface of virus-infected cells, generating antibodies to the extracellular domain of M2 (M2e). In the studies of LAIVs reported thus far, protective roles of M2e antibodies are not the main focus since it is believed that M2e is less abundant and M2e antibodies contribute less to protection than antibodies to the surface HA and NA proteins. Several strategies have been designed to increase M2e antibody responses, including VLP, DNA, peptide, and protein vaccines . M2e is NA is the second most abundant surface glycoprotein that has receptor-destroying enzyme activity. The NA cleaves terminal sialic acid reside bound to viral HA and releases progeny virus particles from infected cells, facilitating the virus spread in a host. NA has long been ignored as vaccine antigens since the NA is less abundant than HA on the virion and NA antibodies do not prevent viral entry into cells. However, recent findings on the diverse roles of NA during virus infection cycle underpin the importance of NA antibodies for protection against influenza viruses. In addition to the classical supportive role for viral egress at the later stage of infection, multiple functions of NA have also been demonstrated. It was shown that the sialidase activity of NA is also important for viral entry at the early stage of infection. In the mucus, viral HAs of influenza virus are captured by mucin proteins that are highly glycosylated, and NA cleaves the sialic acid from the mucins and releases the captured viral particles, permitting the viruses to reach host cells . It was In addition to surface proteins HA, NA, and M2, influenza infection induces antibodies directed to internal structural NP and M1, nonstructural polymerase complexes, NS1, and NEP, and even small accessory proteins such as PB1-F2 and PA-X ,60,61,6212U, intatolimod (TLR-3 agonist), and surf clam microparticles, and also novel immune stimulating materials, such as mushroom mycelia extracts and alpha-galactosyl ceramide [The mucosal surface of respiratory tract is the entry site of influenza viruses and, thus, anti-influenza immunity in this site is very important for preventing the viral infection. LAIVs are administered into the nasal route and primarily infect the upper respiratory tract and stimulate mucosal immune responses. There are distinctions in antibody types and antibody titers between the upper and lower respiratory tracts. The lower respiratory tract contains higher titers of IgG antibodies than IgA antibodies with a ratio of 2.5:1, whereas the upper respiratory tract is dominated by IgA antibodies with an IgG to IgA ratio of 1:3 . It is aceramide ,82,83,84ceramide ,86, suggceramide . Despiteceramide . Those sceramide . T cells can recognize endogenously- and exogenously-derived viral linear peptides generated by cleavage of viral proteins in cells. Cytotoxic CD8+ T lymphocytes (CTLs) recognize and kill virus-infected cells and help in the clearing of infected cells from the host. Harnessing T cell immune responses can overcome the limitations of UIV strategies that rely on antibodies in terms of the potency and breadth of cross-protection. Many studies have shown that the induction of cross-reactive T cell immunity is important for providing broad protection against heterologous influenza viruses and thus has been considered as an attractive strategy to construct UIVs. The importance of T cell immunity for providing broad protection and T cell-based UIV strategies have been well-described in several reviews ,91. ThisStaphylococcus aureus, Salmonella enteritidis, and Yersinia pestis, but also to viruses such as HSV and vaccinia virus, and even to tumors in mice. Surprisingly, the protection was shown to last up to 11 months after vaccination, suggesting the exceptional breadth and duration of nonspecific effects of vaccination. Only a part of molecular mechanisms of trained innate immunity of BCG vaccine has been elucidated, including histone modification by H3K4 trimethylation resulting in the relaxation of chromatin and increased gene transcription of receptor-interacting protein kinase-2 in innate immune cells such as monocytes [In addition to adaptive immune responses, an increasing body of evidence has shown the nonspecific beneficial effects of vaccines on mortality and morbidity following infections with unrelated pathogens, which is prominent when live vaccines are used. Live virus infection stimulates the innate immune system, consisting of monocytes, macrophages, neutrophils, dendritic cells, and NK cells, consequently resulting in cytokine and chemokine production. It has been thought that innate immunity does not possess immunological memory and provides nonspecific protection and induces the same effects upon every exposure to a pathogen. However, recent data have suggested that innate immunity can also be trained and have long-term effects by epigenetic reprogramming of related genes of immune cells . The nononocytes . LAIVs honocytes . In the onocytes . It was As discussed above, LAIV-based strategies present several benefits for developing a broad and potent UIV. The delivery of the whole set of viral antigens induces diverse antibody responses to surface and internal viral proteins. Administered via the nasal route, LAIVs stimulate mucosal immune responses that provide the first defense lines to influenza virus infection. In addition, LAIVs induce robust cell-mediated immune responses, primarily T cell immunity. Thus, the simultaneous induction of antibody responses and T cell responses against various viral proteins greatly enhance the breadth and potency of cross-protection . In spitPersistent and inherent issues regarding the use of LAIVs is biosafety concerns on using live viruses as a vaccine. Although CAIVs have proven safe in humans in extensive experimental and clinical settings, the safety of other LAIVs have yet to be fully elucidated through clinical trials. Moreover, it remains possible that reversion into a virulent strain by unwanted mutations or genetic reassortment with other strains occurs in vaccinated individuals . AnotherThe precise molecular mechanisms of the VAERD have not been elucidated yet, and HA stalk-based vaccines do not demonstrate the VAERD in animals and humans, suggesting that correctly-folded HA stalk antigens induce broadly protective antibodies without any harmful effects. In addition, a study has shown that HA specific antibodies that do not block receptor binding can result in antibody-dependent enhancement (ADE) of viral infectivity, highlighting the need for careful monitoring on potential harmful effects of non- or poorly neutralizing antibodies . These VMost humans acquire influenza immunity either by natural infections or vaccinations during their lifetime and preexisting immunity may impact the magnitude and hierarchy of immune responses to subsequent vaccinations. In particular, preexisting immunity can significantly affect the immune responses elicited by LAIVs that replicate in the respiratory tissues ,121. EarThe mode of action and the breadth and potency of individual correlates of protection required for cross-protection have been well-described . HoweverSeveral studies presented mathematical modeling analysis to demonstrate the benefits of using UIV for controlling seasonal influenza and pandemic influenza at a population level. It has been generally expected that although UIVs do not block viral infection against a given strain, they present superior abilities to reduce the epidemic size, pandemic size, and the antigenic evolution of the viruses over strain-matched vaccines ,128. HowThis section suggests several rational vaccination strategies using LAIVs for the development of broad, potent, and safe UIVs. Provided that strain-specific neutralizing antibody responses toward highly variable HA head do not provide uniform broad protection across influenza viruses, UIV strategies based on LAIVs do not have to include particular sets of HA and NA surface antigens, presenting considerable flexibility for rational designs using LAIVs. Considering that the HA gene of a cold-adapted master donor strain also has attenuating mutations , the useThe discovery of broadly neutralizing antibodies against influenza viruses does not translate directly into the development of broadly protective influenza vaccines. While the antibodies present promising strategies for broad therapeutics against the viruses, the induction of such antibodies by vaccination may be considerably difficult. It has been shown that vaccination induce many different polyclonal antibodies directed to HA stalk, each of which differs in binding breadth, protection potency, and the mode of protection. These points may also hold true for antibodies directed to the other viral antigens, such as NA, M2e, and internal proteins. Furthermore, a concern has also been raised on the unexpected induction of non-functional and harmful antibodies that enhance viral infectivity rather than protecting against the virus, as shown by antibodies to the linear epitope of HA2 region. The delivery of correctly folded antigens can overcome VAERD problem by HA stalk antibodies. However, the use of correctly folded antigens necessarily leads to the induction of polyclonal antibodies that have different characteristics. In addition to HA stalk antibodies, it has been shown that the expression of corrected folded NA antigens is essential for eliciting broadly protective NA antibodies by vaccination. In addition, the induction of both antibody responses and T cell responses are required for providing sufficiently broad and potent cross-protection. For this purpose, prime-boost vaccine regimens using different vaccine types are preferred, mostly in combination of IIVs and LAIVs. Mucosal immunity should also be noted as a critical aspect of LAIVs since mucosal antibodies directed to influenza virus surface proteins have been shown to demonstrate broad protective activities. Several animal studies have shown that LAIVs alone can provide exceptionally broad and potent protection by the induction of multiple immune correlates without neutralizing antibodies, suggesting that LAIVs can serve as a standalone vaccine strategy for a UIV. However, several limitations of LAIVs should also be noted, such as low vaccine effectiveness in adults and the elderly, poorly characterized immune correlates of cross-protection, and the lack of controlled human challenge studies. For clinical relevance, considerable efforts are needed to design rational approaches that are able to efficiently induce cross-protective immune responses by LAIVs.


Escherichia coli, with recurrence of the infection three months later. With this case study, we aim to stress the importance of antimicrobial resistance and how it can preclude an otherwise favorable prognosis.Skull base osteomyelitis (SBO) is a life-threatening condition in patients with underlying comorbidities. Ear infections may spread through normal skull base fissures in this group of patients. However, its diagnosis is frequently delayed due to the unspecific clinical findings at onset, such as headache, with diverse cranial neuropathies later as the disease progresses. We present the case of a patient with otogenic skull base osteomyelitis complicated with retropharyngeal extension, treated with surgical drainage and broad-spectrum antibiotics directed toward extended-spectrum beta-lactamase (ESBL)-producing  Skull base osteomyelitis (SBO) is a rare and life-threatening condition comprising a diagnostic and therapeutic challenge. This condition classically presents in the elderly and diabetic patients with nonspecific symptoms such as headache and cranial nerve palsies, mostly affecting the seventh cranial nerve (CN). Various diseases can mimic SBO, therefore requiring a high index of clinical suspicion ,2. HereiA 69-year-old female with a past medical history of type 2 diabetes, hypertension, and penicillin allergy was referred to our tertiary care center for the management of left chronic otitis media complicated with external otitis and ipsilateral peripheral facial nerve palsy ongoing for the past six months prior to our consult. The infection had been previously treated in the primary care setting with trimethoprim/sulfamethoxazole, erythromycin, and metronidazole. A computed tomography (CT) of the head was performed to rule out necrotizing otitis externa. Our initial management consisted of ciprofloxacin, corticosteroid, and a left ventilation tube insertion, with improvement of symptoms. At our three-month clinic follow-up, she reported progressive intermittent retro-ophthalmic pain with radiation to the occipital region, otalgia, tinnitus, and relapse of the purulent otorrhea.Physical examination revealed a House-Brackmann III left peripheral facial nerve palsy. Otoscopy findings were notable for circumferential edema of the external third of the external auditory canal (EAC), purulent otorrhea, and granulation tissue at the level of the isthmus junction of the EAC; the ventilation tube was still present in the left tympanic membrane. Nasal endoscopy revealed a mass in the nasopharynx medial to the left Eustachian tube. Cranial nerve examination was unremarkable. An initial culture of the otorrhea was sent on admission to the hospital.Escherichia coli in the initial culture. Further laboratory studies are shown in Table She was admitted for diagnostic workup and antibiotic therapy with levofloxacin and clindamycin, which was escalated to meropenem and vancomycin by the infectious disease specialist after antimicrobial susceptibilities resulted with ciprofloxacin-resistant and extended-spectrum beta-lactamase (ESBL)-producing A contrast-enhanced CT scan of the head revealed findings of otitis media, clival demineralization, and a hypodense mass in the preclival region. Complementary magnetic resonance imaging (MRI) revealed a T1 hypointense and T2 hyperintense with peripheral rim enhancement lesion and diffusion restriction in diffusion-weighted imaging (DWI) (Figure An endoscopic approach to the nasopharynx and the left EAC was used for drainage and to obtain biopsy specimens and further tissue cultures Figure . An unspSBO is a rare entity characteristic of immunocompromised patients with nonspecific symptoms in relation to the affected structures . It is cPseudomonas aeruginosa is the most common infectious agent; nevertheless, other Gram-positive bacteria such as Staphylococcus, Streptococcus, or even fungi have been described as etiological agents [Escherichia coli as species produce extraintestinal disease in immunocompromised hosts; this infectious agent can be resistant to ciprofloxacin [Escherichia coli reported in our literature review.Among the causative microorganisms, l agents ,5. Eschefloxacin . Acquirefloxacin . Albeit Early imaging is crucial, as the unspecific clinical findings of this entity can delay diagnosis, and further complications can easily appear . The diaBroad-spectrum antibiotic therapy with antipseudomonal coverage is considered the mainstay of treatment. Current evidence recommends a longer duration of intravenous antibiotics, although this is still somewhat controversial. Regarding surgical management, evidence is still inconclusive, but evidence suggests that early debridement of necrotic tissue improves clinical outcomes and reduces inflammation burden. We opted for a four-week intravenous antibiotic therapy course and outpatient treatment for two more weeks with ciprofloxacin ,9.Our patient presented with recurrent episodes of headache and persisting otorrhea, which prompted further diagnostic workup. Cultures and contrast-enhanced imaging were crucial to the decision-making in this case. There was an absence of bone destruction on the external, middle, or internal ear, which made it more difficult to reach a diagnosis until bone erosion was evident in the clival region. With this case study, we aim to highlight the importance of maintaining a high clinical index of suspicion, obtaining cultures to guide antimicrobial therapy, and prompt imaging studies to guide duration, treatment, and response during the follow-up period.Skull base osteomyelitis is a poorly described clinical entity in our environment. It requires a high clinical suspicion to be diagnosed promptly, reducing morbidity and mortality from complications related to this condition. An accurate diagnosis in a timely fashion is imperative to improve prognosis in patients with SBO. With this report, we aim to contribute to filling in the gaps in knowledge in the decision-making processes of these challenging cases with the goal of improving outcomes in SBO and avoiding recurrence and complications.


The introduction of new therapeutics for patients with chronic heart failure, including sacubitril/valsartan, sodium-glucose cotransporter 2 inhibitors, and ivabradine, in addition to beta-blockers, angiotensin converting enzyme inhibitors, and mineralocorticoid receptor antagonists, lends an opportunity for significant clinical risk reduction compared to what was available just one decade ago. Further clinical options are needed, however, for patients with residual clinical congestion refractory to these therapies. Adaptive servo-ventilation is a novel therapeutic option to address significant clinical volume in cases resistant to medical therapy. The aggregate benefit of these additional therapeutic strategies in addition to foundational medical therapy may be a promising option in the selected candidates who do not achieve acceptable clinical and quality-of-life improvements with oral medical therapy alone. Now is the era to reconsider the implication of an adaptive servo-ventilation-therapy-incorporated medical therapeutic strategy for patients with congestive heart failure. Several novel therapies have been introduced over the last decade that both improve quality of life and reduce mortality in patients with chronic heart failure, including sacubitril/valsartan (ARNI), sodium-glucose cotransporter 2 (SGLT2) inhibitor, and ivabradine . Up-titrIn addition to the survival benefit, improvement in patient-reported outcomes including functional status and quality of life is also of paramount importance . Much ofAdaptive servo-ventilation  is a noninvasive, positive pressure ventilation tool that reduces work of breathing, suppresses sympathetic nervous system activation, and improves pulmonary/systemic congestion via decreasing cardiac preload and afterload in patients with congestive heart failure, if appropriately utilized at least for 4 h during the night  9]. Imp. Imp9]. We believe that ASV therapy can be an effective strategy in managing persistent pulmonary/systemic congestion refractory to medical treatment , and shoAdequate control of congestion is a critically important goal in chronic heart failure management to both reduce mortality and morbidity and improve patients’ symptomology and quality of life . FurtherSacubitril/valsartan was the first of the new medical therapies in patients with chronic heart failure shown to reduce mortality and morbidity compared to the enalapril arm in the PARADIGM-HF trial . HoweverIn the acute phase of decompensated heart failure, early de novo administration of beta-blocker therapy is generally contraindicated given the risk of worsening cardiac output due to negative inotropic effects in the presence of pulmonary congestion . This maThe indications, pressure settings, and timing of termination need to be identified during ASV therapy. For successful ASV therapy and avoidance of congestion, the baseline existence of pulmonary congestion is necessary to be confirmed . InapproTM, Sensible Medical Innovations Ltd., Netanya, Israel) system, which is a noninvasive electromagnetic-based technology to quantify lung fluid volume, has been introduced in the clinical setting , which noninvasively estimates cardiac output  . UnderlyOther therapies may also have the potential to preserve kidney function. Tolvaptan, a vasopressin type-2 receptor antagonist, may have a neutral impact on renal function as opposed to conventional loop diuretics. Tolvaptan has potent aquaretic properties and may decrease the dosage needed of typical loop diuretics, though in some countries this presents a cost challenge . SGLT2 iIn the current era with a new regimen of available medical therapies for heart failure, a combination of ASV and medical therapy may be a promising option for patients with considerable congestion and impaired renal function.


The emergence of single cell technologies provides the opportunity to characterize complex immune/central nervous system cell assemblies in multiple sclerosis (MS) and to study their cell population structures, network activation and dynamics at unprecedented depths. In this review, we summarize the current knowledge of astrocyte subpopulations in MS tissue and discuss the challenges associated with resolving astrocyte heterogeneity with single-nucleus RNA-sequencing (snRNA-seq). We further discuss multiplexed imaging techniques as tools for defining population clusters within a spatial context. Finally, we will provide an outlook on how these technologies may aid in answering unresolved questions in MS, such as the glial phenotypes that drive MS progression and/or neuropathological differences between different clinical MS subtypes. Due to the resounding success of current MS medications in treating relapsing-remitting MS (RRMS), research interest is increasingly focused on disease progression and neurorepair where current treatments are ineffective. In contrast to RRMS, progressive disease is thought be driven by central nervous system (CNS)-intrinsic processes, where infiltration with peripheral immune cells plays only a minor role. Thus, the attention in MS research is arguably shifting from the peripheral immune system to CNS cells. The CNS-intrinsic processes associated with progression include chronic, diffuse activation of glia cells at the rim of chronic active lesions and in the normal appearing white matter (NAWM) , corticaIn this review, we are focusing on astrocytes. Astrocytes are substantially more numerous than microglia cells, accounting for 20–40% of the total glial population, depending on the brain region . In a hoCNS pathologies such as trauma, infection, neurodegeneration, and inflammatory demyelination lead to prominent astrocyte responses with morphological, molecular, and functional remodeling. Reactive astrocytes can promote tissue survival by forming a functional barrier around damaged tissue and contribute to tissue repair . HoweverHere, we review the progress made in identifying astrocyte populations in MS and its mouse model, experimental autoimmune encephalomyelitis (EAE). We will discuss the limitations of current snRNA-seq methods in identifying astrocyte subpopulations and the potential of highly multiplexed imaging in further elucidating astrocyte heterogeneity by adding spatial information.In contrast to oligodendrocytes and microglia, astrocytes are morphologically heterogenous at a resting state. At least nine morphologically distinct astrocyte subpopulations have been identified, of which four subtypes are present within the human neocortex  . AdditioOne of the first efforts to identify astrocyte subpopulations was based on marker expression. Genetically green fluorescent protein (GFP)-labeled astrocytes were isolated and screened for 81 cell surface antigens with fluorescence-activated cell sorting (FACS) . This idin situ hybridization (smFISH), the study found that cortical astrocytes segregated into a superficial, mid and deep layer pattern, which did not match the neuronal layering  approaches use fluorescence-labeled small oligonucleotides to probe single mRNA transcripts. SeqFISH uses multiple rounds of hybridization and has been shown to be scalable to the genome level in vitro . SmFISH in vitro  and to min vitro . A furthin vitro .Additionally, a method termed deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) delivers DNA barcodes to tissue at a specific location via a microfluidics platform and sequences the spatially barcoded mRNA and proteins . Under iHighly multiplexed imaging has been driven by imaging mass cytometry (IMC), a technology that, like CyTOF, uses antibodies conjugated to lanthanide isotopes . Tissue t-distributed stochastic neighbor embedding (tSNE) , segmentation of cell types of interest with workflows such as ilastik pixel classification  and Cellg (tSNE) , as withg (tSNE) ] to locag (tSNE) . Some ofg (tSNE) , which uWe have applied IMC to acute MS lesion tissue to investigate the landscape of myeloid and astrocyte phenotypes in acute and chronic active MS lesions . In thisSingle cell genomics, spatial mRNA profiling and highly multiplexed (protein) imaging offer different advantages and disadvantages. While both single cell genomics and spatial mRNA profiling target the whole transcriptome; RNA-seq is superior in isolated cells/nuclei compared to tissue with regard to gene coverage and sequencing depth. In contrast, highly multiplexed protein imaging has a parametric depth of only dozens to perhaps hundreds of markers, but allows for precise outlining of cells with complex, irregular morphologies such as astrocytes, which will improve the spatial analysis of individual cells. Although it is implicitly assumed that mRNA expression correlates with protein expression, this correlation varies substantially with different gene classes , making With optimized protocols to capture nuclei from astrocytes and other under-represented CNS cell types such as microglia, improvements in single cell genomics and multi-omics integration will allow generation of complete single-cell atlases of healthy CNS and neurological diseases. These atlases will include epigenomic and transcriptomic profiles, subpopulation localization, spatial and functional (receptor-ligand) cellular interactions and pathway annotations. Applied to carefully characterized MS tissue, this will identify the constituent subpopulations and cellular interactomes that are specific to key pathological processes such as acute demyelination, chronic glial activation in progressive MS, and remyelination. This comprehensive approach will identify therapeutic novel targets to ameliorate disease progression and to promote neurorepair.CL, MS, MM, SB, ST, and DP wrote the manuscript. All authors contributed to manuscript revision, 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.


Next-generation sequencing (NGS) has played an important role in detecting genetic variants with pathologic and therapeutic potential. The advantages of NGS, such as high-throughput sequencing capacity and massively parallel sequencing, have a significant impact on realization of genetic profiling in clinical genetic laboratories. These changes have enabled clinicians to execute precision medicine in diagnosis, prognosis, and treatment for patients. However, to adapt targeted gene panels in diagnostic use, analytical validation and ongoing quality control should be implemented and applied with both practical guidelines and appropriate control materials. Several guidelines for NGS quality control recommend usage of control materials such as HapMap cell lines, synthetic DNA fragments, and genetically characterized cell lines; however, specifications or applications of such usage are insufficient to guideline method development. This review focuses on what factors should be considered before control material selection for NGS assay and practical methods of how they could be developed in clinical genetic laboratories. This review also provides the detailed sources of critical information related to control materials. Next-generation sequencing (NGS) has had a drastic impact on clinical genetic laboratories involved in detecting mutations and clonal heterogeneity, therapeutic decision making, monitoring therapy response, and disease prediction for at-risk patients through technical strength ,2,3,4.With the advantages of NGS technology such as massively parallel sequencing, high-throughput sequencing data, and low sequencing cost per base, clinicians are able to provide more rapid diagnoses and patients would receive adequate treatment. However, this only becomes possible under the assurance of complete quality control (QC) similar to other molecular methods. QC of NGS method is more challenging because NGS data is generated through complex protocols . NGS worBecause of these complexities, the College of American Pathologists (CAP) checklist requires specific quality management programs that specify controls, metrics, and quality control parameters to monitor overall procedures in a clinical laboratory . To ensuWe summarized the factors to consider when selecting control materials in Nucleic acids extracted from whole blood, tissue, or cytological specimens such as direct smear, liquid based cytology, or supernatant are usually used for genetic tests. If these samples are not in a low-cellularity status, high-quality DNA or RNA can be extracted . Howeverhttps://www.ncbi.nlm.nih.gov/clinvar, accessed on 21 April 2021), Online Medelian Inheritance in Man , GeneReviews , the Human Gene Mutation Database , Leiden Open Variation Database , Database of genomic variation and Phenotype in Humans using Ensembl Resources , or other locus specific databases, etc. for germline variants and Catalogue Of Somatic Mutations In Cancer , The Cancer Genome Atlas  and International Cancer Genome Consortium (ICGC) Data Portal , etc. for somatic mutations.To develop control materials for a targeted gene panel for a specific disease, prevalent pathogenic variants and their types for the tested genes need to be well characterized and considered. To confirm which variant types compose the pathogenic variants in a specific disease, it is necessary to review databases for genetic variants or related papers. Some examples include ClinVar , insertion and deletions (indels), copy number variant (CNV), and structural variant (SV)) but also the characteristics of variants’ regions  need to be considered . AlthougDue to the diversity of genetic variation including germline variants and somatic mutations in humans, the targeted NGS panel is designed according to the specific target-genes and their genetic characteristics. Panel for germline variants is implemented to detect associated genes with particularly hereditary variants in families ,18. Diplhttps://www.nist.gov/programs-projects/genome-bottle, accessed on 21 April 2021) from National institute of Standards and Technology (NIST) produced high-confidence variant sets for HapMap samples such NA12878, NA24385, NA24631, and made them publicly available. [https://www.nist.gov/programs-projects/genome-bottle, accessed on 21 April 2021) could be compared with experimental NGS data [https://www.biorxiv.org/content/10.1101/2020.07.24.212712v3, accessed on 21 April 2021). Cell lines or DNA for NIST reference samples can be obtained from the Coriell Institute .We summarized control materials for next-generation sequencing in NGS data . RecentlNGS data  and smalhttps://www.cdc.gov/labquality/get-rm/index.html, accessed on 21 April 2021), Coriell Institute Human Genetic Cell Repository , European Collection of Authenticated Cell Cultures  or commercial reference materials (CRMs) such as SeraCare  or Horizon Diagnostics  are helpful in getting the information of reference materials for specific hereditary genetic disorders as well as human leukocyte antigen testing or pharmacogenetics. Although there are various information sources and reference materials for hereditary genetic diseases, it is still challenging to get adequate reference materials for rare diseases, as multiplexing cell line-based reference materials to include the variety of clinically relevant pathogenic variants continuously dilutes VAF. Genetic variants detected in patients’ samples that are validated by orthogonal molecular methods such as Sanger sequencing, multiplex ligation-dependent probe amplification, or quantitative polymerase chain reaction (PCR) can also be used as means of quality control. However real patients’ samples are finite in their amount compared to cell lines; therefore, both cell lines and real specimens are used together to validate the clinical performance of customized targeted panels and only cell lines are sequenced for ongoing QC in routine clinical runs [Validation process for detecting germline variants in certain hereditary diseases or conditions should include samples with representing pathogenic variants. Genetic Testing Reference Materials Coordination Program from Centers for Disease Control and Prevention  or Cancer Cell Line Encyclopedia (CCLE) . Cancer cell lines can be obtained through American Type Culture Collection , or DSMZ . However, true VAF of heterozygous variants in cancer cell lines is unclear because it may be influenced by genomic instability and drift.In a clinical laboratory, cancer cell lines or CRMs are usually used for validation and/or QC for detecting somatic variants due to ease of access and applicability. Somatic mutations in cancer cell lines are well-defined in COSMIC , and SeraCare  are specifically designed using gene editing and gene modulation. The CRMs include somatic hotspot mutations which allele frequencies are validated by absolute quantification method. However, CRMs are not cost-effective to use for continuous QC in routine clinical runs.The CRMs for NGS that are accessible at Horizon Diagnostics  or the study by Craig DW et al. [In-silico data sets can be used to validate bioinformatic analysis: some example studies include benchmark somatic variant calls and regions from the study by Lee AY et al.  , next selecting well-characterized cancer cell lines that contain expected mutations in databases , after that, quantifying the allele frequency of major concerned variants included in selected cancer cell lines by digital PCR, then, calculating the correct ratio of the cell lines to mix, culturing and extracting nucleic acids from the selected cancer cell line, diluting of cancer cell line to HapMap cell line to make desired allele frequency (set the VAF of hotspot mutations as low as the LOD level), then mimicking FFPE or cfDNA if necessary, quantifying the allele frequency of expected major concerned variants in developed control material by digital PCR, and finally, establishing an answer set (VAF range for detected non-hotspot and hotspot mutations) through NGS assay. We simplified the schematic diagrams of developing multiplexed reference materials to detect somatic mutations in In order to include the maximum number of minor concerned variants with various allele frequency ranges, it is recommended to consider the following: increasing the ratio of cell lines that have fewer mutations, leads of reduction of ratio of cell lines that have more homozygous mutations . It is aFor mimicking FFPE specimen, cell line pellet needs to be fixed and embedded by formalin and paraffin as patient’s tissue specimen is processed . A previSynthetic DNA  is another option to supplement the use of multiplexing cell lines for control material ,39,40. AThe reference materials using engineered cell line by gene-editing  is also another option . They inIn this review, we have discussed the considerations to select adequate control materials for NGS assay, the type of QC materials, and how they could be developed in clinical laboratories. Although several previous review studies have discussed the types of control materials for NGS and their advantages and disadvantages, however, in this review, we focused on practical aspects of selecting and developing NGS QC materials in clinical genetic laboratories, explaining what we consider and need to understand, so that they can be applied directly it in their clinical laboratories. In addition, because the sources of critical information for the development of NGS QC are scattered, it is difficult to obtain such information in genetic laboratories that do not have sufficient experience, so we intended to describe it as much detail as possible. Although assay design and validation are performed according to the standard guidelines in clinics, screening of systemic errors with adequate control materials that potentially occur in routine runs is highlighted. To verify the entire process of NGS assay for accuracy, precision, analytical sensitivity, analytical specificity, and the LOD, this review has introduced several options for control materials. Each control material has pros and cons. Clinical laboratories should develop quality management programs according to the purpose and characteristics of NGS assay.


Plants produce numerous structurally and functionally diverse signaling metabolites, yet only relatively small fractions of which have been discovered. Multi-omics has greatly expedited the discovery as evidenced by increasing recent works reporting new plant signaling molecules and relevant functions via integrated multi-omics techniques. The effective application of multi-omics tools is the key to uncovering unknown plant signaling molecules. This review covers the features of multi-omics in the context of plant signaling metabolite discovery, highlighting how multi-omics addresses relevant aspects of the challenges as follows: (a) unknown functions of known metabolites; (b) unknown metabolites with known functions; (c) unknown metabolites and unknown functions. Based on the problem-oriented overview of the theoretical and application aspects of multi-omics, current limitations and future development of multi-omics in discovering plant signaling metabolites are also discussed. Small molecules produced by plants play vastly diverse roles in nature, amongst which signaling and communication are two of the most important aspects. Plant metabolites are broadly classified into primary and secondary metabolites. Primary metabolites are ubiquitous to all plants whereas secondary metabolites are specifically produced by certain plants, tissues and cells and in most cases elicited under certain conditions. It is estimated that there are over one million metabolites produced throughout the plant kingdom . SecondaArabidopsis thaliana [A. thaliana root length, respectively. Defense compound glucosinolate can also influence plant growth via its degradation product indole-3-carbinol, which inhibits root elongation by competing directly with auxin as a signaling molecule, so as to maintain the balance between plant growth and plant defense [Oxalobacteraceae [These metabolites can serve as signaling molecules during plant growth and development, initiating and coordinating plant developmental programs. In the meantime, they can “liaise” with external environments and other living organisms, fulfilling the subtle demands for plant health and growth. Plant hormones including jasmonic acid , abscisithaliana  were fou defense . Other pteraceae . Such interaceae .The aforementioned examples demonstrate that even some of the best-known plant metabolites still have unknown functions awaiting discovery. Moreover, the majority of plant metabolites discovered so far have only been chemically/structurally characterized and not yet been assigned a definite function in nature. The plant metabolites that we have already discovered might actually represent only the tip of the iceberg regarding the metabolic diversity of plants, as implicated by the numerous uncharacterized predicted biosynthetic genes present in plant genomes . CurrentThere are a few major challenges impeding the discovery of plant signaling molecules: (i) the content of plant signaling metabolites are usually very low; (ii) plant signaling metabolites are often under dynamic metabolism ; (iii) plant signaling metabolites normally have characteristic spatial–temporal distributions ; (iv) they have extremely diverse physical and chemical properties that demand customized analytical and assay methods; (v) they have diverse specialized functions that can only be captured under specific spatial and temporal conditions.Addressing these challenges requires interdisciplinary approaches. Multi-omics is a powerful and indispensable integrated technique that has greatly accelerated the discovery of plant signaling metabolites via systematic comparative analysis of large datasets . ExperimBiological networks are highly complex, interconnected and tightly regulated. Plant metabolites are the output of the Central Dogma, closely related to phenotypes and associated with various aspects of cellular processes ranging from biosynthesis and catabolism to regulation, transport, mode of action and their interactions with environmental changes. Each of the related aspects provides an entry point for investigating plant signaling metabolites. These entry points correspond well with the different levels of omics  that are currently available. Different levels of omics techniques will have to be employed in a combinatorial fashion to reveal a relatively complete picture of plant signaling metabolites. Depending on the nature of the study, and the current knowledge of the structures and functions of the metabolites of interest, one might design experiments with specific focus on one or two omics techniques. Nevertheless, an in-depth grasp of the technical features and research aims are critical for the successful execution of multi-omics.Multi-omics refers to the integrated application of more than one type of large dataset analysis, including genomics, epigenomics, transcriptomics, proteomics, metabolomics and microbiomics. To better understand biological activities at a system level, traditional single-omics research is rarely comprehensive enough and requires the integrated multi-omics data for global analysis of biological systems , and mulAmaranthaceae, Fabaceae, Rosaceae and Solanaceae families [Genomics involves the study of complete DNA sets in organisms, including all its genes, their sequences, arrangements and architecture, providing perspective for looking into biological problems from the most basic code of life DNA. DNA carries instructions for transcription , translation (start and stop codons) and specific functions of a gene (coding sequence) . Genomicfamilies . With thfamilies , phytoclfamilies  and clusfamilies ,27 have DNA in cells is wrapped around histone proteins H1, H2A, H2B, H3 and H4, which form spool-like structures that enable very long DNA molecules to be wrapped up neatly into chromosomes inside the cell nucleus. DNA and histones can undergo reversible chemical modifications like DNA methylation or histone methylation, acetylation, phosphorylation and adenylation, the complete set of which in cells are heritable without changing the DNA sequence, termed epigenome. Epigenomics unitizes high-throughput technologies to decipher epigenome landscapes based on comprehensive analyses.Epigenome landscapes are tightly associated with gene activity and expression, controlling the production of proteins and metabolites under a specific condition via altering chromatin conformation or transcription regulator recruitment. DNA methylation is one well-known epigenomic process with methyl groups being added to the bases of a DNA molecule at specific sites, switching the genes on or off by altering interactions between the DNA and methyl group reading proteins. Epigenomics technologies including chromatin immunoprecipitation–sequencing (ChIP–seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC–Seq)  enable dApart from plants having complete genome sequences as mentioned above, epigenomics may also be applied to decouple the regulatory mechanisms underlying plant signaling metabolite biosynthesis in non-model plants that lack a whole genome, using techniques such as epiGBS, reference-free reduced representation bisulfite sequencing  for explTranscriptomics is used to study all types of RNA transcripts including messenger RNAs (mRNAs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) present in a sample under specific conditions. As one of the most widely used high-throughput sequencing methods, modern transcriptomics technology has developed from bulk RNA sequencing (RNA-seq) at the tissue or population level to single-cell RNA-seq at the individual cell level using nanopore sequencing and 10× Genomics single-cell sequencing . In contTranscriptomics is particularly useful when no genome information is available for the plant to be studied, as RNA-seq data can be assembled de novo to retrieve coding sequences (CDS) of biosynthetic genes. This has proven to be a powerful tool for discovering biosynthetic genes responsible for the synthesis of metabolites  , gas chrOxalobacteraceae, which aided maize in acquiring nitrogen under nitrogen deprivation [Recent studies have proven that the root microbiome, modulated by plant signal metabolites like coumarins, flavones and benzoxazinoids, improves plant stress resilience . Microbirivation .The different features of each single omics mentioned above can be synergistically. oriented for discovering signaling metabolites, in terms of both structures and functions ,67,68,69Burkholderiaceae strains in iron-limiting soil. Through further root transcriptional profiles and elemental analysis of Col-0 and coumarin-deficient mutant f6′h1 plants under available iron or the unavailable form of FeCl3 media with live or heat-killed synthetic community (SynCom), the role of coumarins, especially fraxetin, in mediating root microbiota for improving plant performance under iron-limiting conditions, was uncovered [The integration of multi-omics analysis into studies designed to uncover the metabolic basis of certain phenotypes or traits could lead to the discovery of new functions for some well-known molecules. Coumarins, a family of benzopyrones , well-known for their defensive role in nature , were rencovered   mutant that is deficient in systemic acquired resistance (SAR) with wild type Arabidopsis plants. Although FMO1 has been identified as a key component in mediating the SAR against pathogens for Arabidopsis [Pseudomonas syringae treatment but absent from all fmo1 mutant plants. Further structural elucidation based on mass spectra fragmentation and synthetic standards confirmed the chemical identity of the mass signal as glycosylated N-hydroxy-pipecolic acid, suggesting that FMO could hydroxylate pipecolic acid to form N-hydroxy-pipecolic acid, which can be further glycosylated in planta [Arabidopsis mutant npr1  with reduced salicylic acid content and the snc2  mutant which displays an autoimmune phenotype with an excess of salicylic acid were used to perform comparative untargeted metabolomics analysis, which successfully identified new intermediaries for salicylic acid biosynthesis [An untargeted metabolomics approach is essential to uncover novel molecules that might have given rise to certain biological functions. The comparative metabolic profiling of samples with and without biological activities can capture the chemical differences in these samples unbiasedly, enabling the design of experiments to further investigate the structures and biological activities of these chemicals. Many previously unknown molecules have recently been identified using an untargeted metabolomics approach ,77,84,85bidopsis   relative low cost of RNA-seq sequencing and ease of transcriptome assembly as compared to genome sequencing and assembly; (ii) amplifiable signals of transcriptomic changes can be captured with high accuracy and relatively low amounts of plant materials in contrast to the relatively large quantity required for untargeted metabolomics analysis; (iii) functional annotation of transcriptomics sequences with higher prediction accuracy in comparison to untargeted metabolomics analysis. The new cyanogenic metabolite in analysis .Manduca sexta fed with tobacco plants containing normal and compromised diterpene glycoside levels as well as its frass [Manduca sexta fed with tobacco plants containing normal diterpene glycoside levels accumulated significantly more long chain bases, which are substrates of ceramide synthase inhibited by the diterpene glycosides, than those fed with compromised levels of the diterpene glycosides. Moreover, the frass of M. sexta fed with tobacco containing the normal level of diterpene glycosides also accumulated more modified 17-HGL-DTGs than those fed with tobacco containing compromised levels of diterpene glycosides. The identification of modified 17-HGL-DTGs as novel compounds and their activities in inhibiting ceramide synthase led to the further discovery of the toxicity of modified diterpenes  on tobacco plants. It is interesting to note that the ceramide synthase inhibition activity of 17-HGL-DTGs on M. sexta was identified based on cues obtained from comparative transcriptomics analysis of wild type tobacco and autotoxic tobacco mutant plants [It is clear that untargeted metabolomics analysis has to be carried out to correlate with transcriptomics data for the identification of structurally unknown compounds. In some cases, untargeted metabolomics analysis on the organism  interacting with plants can also provide cues leading to the identification of unknown metabolites with novel functions ,89. In ats frass ,89. Mandt plants  (Figure Multi-omics technology has greatly facilitated the discovery of plant signaling metabolites in many aspects; however, technical limitations in individual omics techniques still pose challenges to their application. With regard to genomics, although long-read sequencing such as PacBio  and NanoSimilarly, sequencing read length and accuracy also affect de novo transcriptome assembly, functional annotation, gene cloning and functional validation, especially for those plant species without a sequenced genome. Currently, RNA-seq data are primarily generated using second-generation Illumina sequencing due to the low cost and relatively well-developed analysis pipeline . Single-Although single-cell RNA sequencing techniques have greatly expanded multi-omics application, yielding hidden and more complete mechanistic insights, the development of single-cell metabolomics, in comparison, still lags far behind. This is primarily due to the fact that metabolite signals could not be amplified the same way as DNA and RNA and instrument sensitivity is not yet up to the point of detecting comprehensive sets of metabolites within a single cell . MS-baseIt is foreseeable that the discovery of plant signaling molecules will accelerate in the near future with the increasing availability of omics tools. Novel entities and functions of plant signaling molecules at single-cell or cell-type levels will be an important research direction going forward. In addition, the discovery of plant signaling molecules involved in the interaction between plants and environments or other living organisms will also be a trend in the field with future research. With a better understanding of the functions of plant signaling molecules, their utility will be further exploited, increasing the potential of commercialization, especially in agriculture-related areas. This will also fuel the development of sustainable production technologies including synthetic biology.


Create a prioritization framework for value-based improvement in neonatal care.A retrospective cohort study of very low birth weight (<1500 g) and/or very preterm (<32 weeks) infants discharged between 2012 and 2019 using the Pediatric Health Information System Database. Resource use was compared across hospitals and adjusted for patient-level differences. A prioritization score was created combining cost, patient exposure, and inter-hospital variability to rank resource categories.Resource categories with the greatest cost, patient exposure, and inter-hospital variability were parenteral nutrition, hematology (lab testing), and anticoagulation , respectively. Based on our prioritization score, parenteral nutrition was identified as the highest priority overall.We report the development of a prioritization score for potential value-based improvement in neonatal care. Our findings suggest that parenteral nutrition, central venous access, and high-volume laboratory and imaging modalities should be priorities for future comparative effectiveness and quality improvement efforts. As national healthcare expenditures have grown, increasing attention has been paid to value  across medical fields, including neonatology , 2. IdenUnwarranted practice variation has been identified as a key contributor to health care inefficiency and waste , 6. PrioTherefore, our objectives were to estimate the inter-hospital variability of clinician-driven tests and treatments (CTTs) among very low birth weight and very preterm infants during their birth hospitalization, and create an objective prioritization framework for value-based improvements in neonatal care by combining data on cost, use and inter-hospital practice variation.We conducted a retrospective cohort study of very low birth weight  and very preterm  <32 weeks) infants admitted to neonatal intensive care units (NICUs) in the United States (US) children’s hospitals affiliated with the pediatric health information system (PHIS) database and discharged from 2012 to 2019, to estimate the cost and inter-hospital variability of CTTs ordered during hospitalization.International Classification of Diseases, Ninth and Tenth Revision, Clinical Modification ), and daily resource utilization. Resources at each hospital are mapped to a common set of clinical transaction codes which are organized into imaging studies, clinical services, laboratory tests, pharmacy, supplies, and other  charges. IBM Watson Health  manages the data warehouse function for the database. Data are subjected to reliability and validity checks and must pass a specified threshold of quality before being incorporated into the database. All personal health information is deidentified within the database. A protocol was reviewed by the Baylor College of Medicine Institutional Review Board and was not considered human subjects research.PHIS is an administrative database containing hospitalization data from 51 tertiary-care children’s hospitals, maintained by the Children’s Hospital Association . The database contains data on demographics, diagnosis and procedure codes (using Subjects were identified as either VLBW or VP by discrete data for GA and birth weight (BW), or by diagnostic code if discrete data were unavailable. Subjects less than 22 weeks’ gestation or <400 g were excluded. Subjects admitted after 1 day of age were excluded since we were unable to measure resource use at the referring hospitals. Utilization and cost were only considered for days with a NICU bed charge to exclude costs incurred in other units . We excluded subjects with congenital anomalies that could significantly impact care costs using ICD-9/10 diagnosis codes. Two authors  reviewed all ICD-9/10 diagnosis codes among the potential cohort and independently assigned codes for exclusion. Disagreements were settled by consensus. Excluded diagnoses included critical congenital heart defects and congenital malformations of other organ systems . Diagnosis codes for a patent ductus arteriosus and atrial septal defects, common diagnoses among preterm infants, were not excluded. To account for potential data entry errors, we utilized a number of other exclusions which have been previously described . In addiPharmaceutical, laboratory, and imaging billing were classified into clinically relevant CTT categories  and costs were estimated, as previously described . In brieWe assessed patient-level severity of illness using a length-of-stay-based relative weight approach. We adapted the Hospitalization Resource Intensity Score for iKds (H-RISK) by restricting the all-patient refined diagnosis-related groups  to only those admitted to PHIS NICUs and by basing our calculation of relative weights on NICU days  These ret-test for normally-distributed continuous variables. Multivariable generalized linear mixed models (GLMM) were used to identify demographic and clinical characteristics associated with overall CTT-related spending and the odds of having a resource-intensive NICU day. Covariates in the model were the demographics listed above and our calculated NICU-SOI score. All GLMMs included a random hospital effect to account for the clustering of NICU patients at the same hospital.We summarized categorical variables using frequencies and percentages, non-normally distributed continuous variables using medians and interquartile range (IQR), and normally distributed variables using mean and standard error (SE). Demographics in PHIS include sex, GA, birth weight, age at admission, admission source, race/ethnicity, insurance type, median household income, and disposition at hospital discharge . We compared demographics and the NICU-SOI score between resource-intense NICU stays and resource mild/moderate NICU stays using a chi-square test for the association for categorical variables, a Wilcoxon rank-sum test for non-normally distributed continuous variables, and a two-sample We assessed hospital-to-hospital variation in total CTT-related costs by estimating the intraclass correlation coefficient (ICC) as the percentage of total variation attributable to hospital variation after adjusting for patient demographics and our NICU-SOI. Race/ethnicity was included as a variable because of its association with quality of care. We idenNext, we created a variability index that estimates hospital-to-hospital variation in utilization and allows for direct comparisons across CTT categories. The variability index was derived to capture two types of inter-hospital variation; variation in the proportion of patients exposed to a given CTT category at least once (“exposure variability”), and variation in utilization of the CTT category among those exposed (“utilization variability”). Exposure variability was calculated using the adjusted percent exposure in the entire cohort  and the measured spread of adjusted hospital-specific exposures around the mean using standard distances. Exposure variability was estimated by calculating the standard deviation of those standard distances. This approach estimates variability in the proportion of infants exposed for each CTT category at each hospital beyond what would be expected to occur by chance when sampling from the overall population. Utilization variability was calculated using CTT-related costs, which act as a surrogate for utilization in this case because the PHIS database applies an average cost estimate to all patients across included hospitals. Higher costs on a given hospital day  mean more utilization of that CTT category. Utilization Variability was estimated by calculating the coefficient of variation (CV) of the adjusted hospital mean total costs among exposed patients. The variability index for a CTT category was then calculated as the standardized Euclidean distance of the exposure and utilization variabilities described above. To minimize the impact of the hospital to hospital variation in exposure due to differences in billing patterns , we excluded hospitals from an individual CTT category variability index calculation if their hospital exposure rate was more than four times or less than one-quarter of the overall population exposure rate for a given CTT category.P-values less than 0.05 were considered statistically significant. All data management and analyses were conducted using SAS v9.4 .Lastly, we developed an overall prioritization score for each CTT category by calculating the standardized Euclidean distance (from the origin) based on three factors; total adjusted costs, the proportion of patients exposed, and variability. A flowsheet of the methodology to create the prioritization score is included in the online supplement  are represented by a bubble chart that plots the inter-hospital variability index by the total cost Fig. . The topWe report the first value-based prioritization framework for comparative effectiveness research and quality improvement initiatives in the care of preterm infants. Combining estimates of cost, exposure, and inter-hospital variability into a single prioritization score, we ranked test and treatment categories to identify targets for further research with the highest potential for improving the value of neonatal care. Among a cohort of 26,098 infants across 8 years, the top 10 high priority CTT categories were responsible for $185,820,182 in costs , and include many commonly used tests and treatments in neonatal care, suggesting value-based improvements should focus on optimizing our approach to routine neonatal care. The use of parenteral nutrition was identified as the highest priority overall, followed by anticoagulants , and a number of commonly used laboratory test categories .Estimates of variability have been used previously to prioritize value-driven efforts. Lee et al. measured cost variability to prioritize a value-driven outcomes program and found prematurity had the third-highest variation indirect costs among inpatient and outpatient diagnoses, highlighting the importance of focusing on variability in preterm infant care . Keren ePractices identified by our prioritization score may represent potential opportunities for targeted deimplementation of routine tests and treatments. Deimplementation science is the process of identifying low-value services that can be safely eliminated or reduced in practice , 27. WhiWhile our prioritization framework focuses on cost and variability, another important factor when setting priorities is the reduction of unnecessary harm. Direct harm, such as side effects that result from medications, are easier to identify through traditional methods of study. However, indirect harms from unnecessary testing can also be significant but are generally more difficult to identify. The cascades of care have been described in other specialties in which unnecessary testing leads to further wasteful and harmful downstream care pathways –33. In aOur study has several limitations. The PHIS database has a data quality control program to minimize the risk of data errors. We further minimized the risk of misclassification by applying exclusion criteria to improve data quality. However, using billing patterns to estimate variation in utilization may partly reflect differences in coding systems between hospitals. The PHIS database robustly combines different hospital billing definitions into a unified coding system and we further reduced miscoding risk by excluding extreme outlier hospitals, which may represent differences in billing practices that were unaccounted for. Cost-to-charge ratios are a commonly used tool to estimate cost, used by many large administrative databases like the National Inpatient Sample and the Kids Inpatient Database, and many economic evaluations –40. DespThere are also important limitations to the generalizability of these findings. The PHIS database is comprised of freestanding US children’s hospitals, which may not fully reflect practice patterns in other settings such as community birth hospitals where preterm infants often receive care. Higher-level neonatal units  may spend more on patient care, even after adjusting for GA, outborn status, and patient mix . This coWe established a value-based prioritization framework for comparative effectiveness research and quality improvement based on cost, inter-hospital variability, and degree of exposure to different tests and treatment categories among VP and VLBW infants cared for in US children’s hospitals NICUs. We identified parenteral nutrition, anticoagulation, intravenous fluids, and frequently used laboratory and imaging modalities as top priorities for comparative effectiveness research and quality improvement efforts to increase the value of neonatal care.Supplemental Index and TablesSupplemental Figure 1Supplemental Figure 2


Right ventricular (RV) systolic function represents an important independent predictor of adverse outcomes in many cardiovascular (CV) diseases. However, conventional parameters of RV systolic function (tricuspid annular plane excursion (TAPSE), RV myocardial performance index (MPI), and fractional area change (FAC)) are not always able to detect subtle changes in RV function. New evidence indicates a significantly higher predictive value of RV longitudinal strain (LS) over conventional parameters. RVLS showed higher sensitivity and specificity in the detection of RV dysfunction in the absence of RV dilatation, apparent wall motion abnormalities, and reduced global RV systolic function. Additionally, RVLS represents a significant and independent predictor of adverse outcomes in patients with dilated cardiomyopathy (CMP), hypertrophic CMP, arrhythmogenic RV CMP, and amyloidosis, but also in patients with connective tissue diseases and patients with coronary artery disease. Due to its availability, echocardiography remains the main imaging tool for RVLS assessment, but cardiac magnetic resonance (CMR) also represents an important additional imaging tool in RVLG assessment. The findings from the large studies support the routine evaluation of RVLS in the majority of CV patients, but this has still not been adopted in daily clinical practice. This clinical review aims to summarize the significance and predictive value of RVLS in patients with different types of cardiomyopathies, tissue connective diseases, and coronary artery disease. The assessment of right ventricular (RV) function may be challenging due to the anatomical shape and position of the RV. A large part of the RV is difficult to visualize by echocardiographic examination, particularly for two-dimensional echocardiography. Several echocardiographic views are necessary to assess RV structure and function, which significantly limits the feasibility of complete evaluation of global and regional contractile function. The apical 4-chamber echocardiographic view is the most used window that allows the assessment of several important quantitative parameters .RV systolic function was proven to be an important independent predictor of adverse outcomes in a large number of cardiovascular (CV) diseases and new evidence indicated a higher predictive value of RV longitudinal strain (LS) over conventional parameters of RV systolic function (tricuspid annular plane excursion (TAPSE), systolic flow velocity across the lateral segment of tricuspid annulus obtained by tissue Doppler (s’), RV myocardial performance index (MPI), and fractional area change (FAC)) ,3,4. RV This review article aims to provide a clinical summary of the importance and predictive value of RVLS in patients with different cardiomyopathies, tissue connective diseases, and coronary artery disease.RV systolic function has a very important role in patients with CMPs. TAPSE, s’, and FAC have been long used as the only parameters of RV systolic function and they demonstrated a significant predictive value in patients with dilated, hypertrophic, and arrhythmogenic CMP, as well as in cardiac amyloidosis. The introduction of speckle tracking echocardiographic imaging significantly changed the perception of RV systolic function evaluation and particularly when studies showed its advantages in the prediction of adverse outcomes in these patients. Echocardiography has a pivotal role in the RV assessment and particularly in evaluation of RVLS. However, due to its possibility for accurate estimation of RV volumes, RV ejection fraction (RVEF), as well as left ventricular (LV) tissue characterization, CMR became more utilized over the last decade. The initial investigations using echocardiography-derived RVLS showed significantly lower values of RV global LS and free-wall LS in patients with idiopathic DCM, whereas TAPSE, s’, RV MPI, and FAC could not identify any difference between idiopathic and ischemic DCM patients . A smallSeo et al. involved 143 patients with DCM in sinus rhythm and followed them for 40 months and showed that there was no difference in the conventional parameters of LV and RV function between patients who experience primary endpoint and those who did not . The priThere is lack of agreement about the predictive value of RVLS in DCM. Monaghetti et al. investigated 208 patients with DCM and none of the RV systolic function parameters  was an independent predictor of composite outcome  . The onlCMR analyses reported a significant prognostic impact of RVLS in patients with DCM. The investigation that involved 192 patients with non-ischemic DCM who were followed for 5 years showed that RVLS ≥ −8.5% was independently associated with the composite outcome (major adverse cardiac events (MACEs), including CV death and cardiac transplantation) . The larRV involvement in patients with hypertrophic cardiomyopathy (HCM) consists of structural and functional changes. The incidence of RV involvement in HCM patients varies significantly depending on the criteria and the method used for the assessment of RV structure and function. The histopathological characteristics of HCM are general myocyte hypertrophy, regions of myocyte disarray, different types of fibrosis, and small-vessel disease ,19. In tD’Andrea et al. used RVLS to distinguish HCM patients from professional athletes and suggested a cut-off value for RVLS of >−16% . The samThe only echocardiographic study that investigated the predictive value of RVLS in HCM patients included 267 HCM patients and reported that patients with RV global LS ≥ −20% and those with RV free-wall LS > −19% had significantly higher mortality during 6.7-year follow-up . MultivaCMR study showed that RV longitudinal, circumferential, and radial strains were significantly lower in HCM patients than in controls . All typSeo et al. performed echocardiographic and CMR studies in 256 HCM patients and provided a 3-year follow-up . The autYang et al. included 384 HCM patients and 150 healthy volunteers and showed that RV LS was an independent prognostic factor for the primary and secondary endpoints . The priTraditional echocardiographic parameters for assessment of RV systolic function such as TAPSE, FAC, and s’ are non-specific and could not provide reliable information about fibro-fatty replacement and kinetic disturbances that are characteristics of arrhythmogenic right ventricular cardiomyopathy (ARVC). RVLS seems to be a reliable measurement of impaired wall motion and has important diagnostic value in the absence of RV dilatation, visual wall motion abnormalities, and a reduced RV systolic function measured by conventional parameters. A cut-off value of −18% for RVLS has been proposed to discriminate normal from abnormal RV segments for diagnosing ARVC patients with high diagnostic accuracy . Figure One of the first studies showed significantly decreased RVLS in asymptomatic carriers and ARVC patients with arrhythmia (ventricular tachycardia and fibrillation), whereas FAC was reduced only in symptomatic ARVC patients . MultivaA recent study showed that echocardiography-derived RV global LS was associated with the risk of RV structural progression during a 5-year follow-up . PatientPieles et al. investigated 120 adolescents with suspected ARVC and demonstrated that patients with definite, borderline, and possible ARVC had significantly lower RVLS than in controls . MultivaThe use of CMR-derived RVLS could help in the differentiation between ARVC patients, borderline ARVC, and those with a positive family history of ARVC . RV circA recent study compared patients with ARVC, RVOT arrhythmia, and Brugada syndrome and found that multidirectional strain  could be used to distinguish these three groups of patients . It is oChen et al. recently reported that CMR-derived RV global LS, circumferential, and radial strains were significantly lower in ARVC patients with LVEF < 55% than in those with LVEF > 55%, even though there was no difference in RVEF between these groups . One of Amyloidosis considers the extracellular deposition of insoluble fibrils that consists of different serum proteins (amyloid). There are several types of amyloid proteins that may affect this process: light-chain (LC) immunoglobulin, mutant hereditary transthyretin (TTR), wild-type TTR, mutant apolipoprotein AI, amyloid atrial natriuretic peptide localized to the atrium, fibrinogen alpha type, and serum amyloid A protein. The presence of cardiac amyloid deposits differs with the type of amyloidosis: wild-type amyloidosis and hereditary TTR-related amyloidosis (ATTR) are characterized by a high percentage of cardiac involvement, whereas cardiac involvement is present in about 50% of cases of patients with LC amyloidosis (AL). RV involvement in ATTR is very frequent and previous studies have showed that TAPSE < 14 mm was an independent predictor of MACE defined as death, heart transplantation, and acute heart failure during the 8-month follow-up period . The echThe largest amount of data regarding the prognostic value of RVLS are generated from studies regarding AL. Cappelli et al. investigated patients with AL amyloidosis with and without cardiac involvement that was defined by LV wall thickness >12 mm and reported significantly lower values of TAPSE, s’, and RVLS in patients with cardiac involvement . UnivariA small study provided a 5.5-year follow-up of 26 AL patients who underwent autologous hematopoietic stem cell transplantation and showed that baseline LV, RV free-wall, and LA longitudinal strains were predictors of overall survival among these patients after stem cell transplantation . The redCMR studies provided valuable information regarding RV mechanics and its their influence on outcome in AL patients. Li et al. involved 87 AL patients with a 21-month follow-up and reported a significant reduction in RV longitudinal, circumferential, and radial strains in AL patients compared with controls . MoreoveConnective tissue diseases are associated with autoimmune response, vasculopathy, and fibrosis of the skin and internal organs. This is particularly valid for systemic sclerosis (SSc), which is related to high mortality due to cardiac and pulmonary affection. Vasospasms of smaller vessels and luminal narrowing induce hypoxia and reperfusion injury, which further promotes fibrosis. Conventional echocardiographic parameters are usually not sensitive enough to detect subtle RV changes in patients with tissue connective disease and therefore speckle-tracking technique has a great value to identify subclinical changes before the manifestation of RV failure.Our study group recently showed that conventional parameters of RV systolic function (TAPSE and FAC) were not different between SSc patients and healthy controls, but 3D RVEF and RVLS, global and free-wall, were significantly lower in SSc patients . MoreoveThe effect of specific therapy for pulmonary hypertension in SSc patients can be also detected by speckle tracking. Mecurio et al. showed that 36-week therapy with ambrisentan and sildenafil in SSc patients with pulmonary hypertension significantly improved RVLS, mainly due to improvement of basal and midventricular segments . The invOther reports did not show the predictive value of RVLS in SSc patients. Tennøe et al. reported that only TAPSE, but not RVLS, was an independent predictor of mortality in SSc patients during a 3.3-year follow-up period . One-milData about the involvement of RV and particularly the role of speckle tracking in patients with systemic lupus erythematosus (SLE) are scarce. Leal et al. reported a significant deterioration in RVLS among adolescents with SLE . SimilarA recent meta-analysis that summarized findings from the previously mentioned three studies showed significantly reduced RVLS in SLE patients, as well as a significant reduction in LV multidirectional strain  in patients with SLE .CMR study investigated 47 women with SLE and found that only RV circumferential strain and not RVLS could discriminate SLE patients with normal RVEF (>50%) from those patients with reduced RVEF (<50%) . HoweverThe available data about RV involvement in patients with coronary artery disease are limited in comparison with information that we have about the LV role. The recent study showed that TAPSE, FAC, and s’ were independent predictors of MACE in both STEMI and NSTEMI patients in the first 30 days after myocardial infarction . The thrThe large study that included 621 AMI patients treated with PCI showed that TAPSE, FAC, and RV free-wall LS were all predictors of worse outcomes during the 2-year follow-up period . After mThe only CMR study that investigated the predictive value of RVLS included 1235 AMI patients who were treated with PCI demonstrated that RV free-wall LS was an independent predictor of outcome  in addition to age, Killip class, and LVLS, whereas RV ischemia was not independently associated with MASE during 1-year follow-up . The redThe prognostic importance of RVLS has been well established in patients with congenital heart diseases. Recently published data showed that free-wall RVLS (> −17%) was the only independent predictor of low functional capacity in patients with repaired tetralogy of Fallot . Study tMoceri et al. investigated patients with pulmonary hypertension associated with congenital heart disease with and without Eisenmenger syndrome and found that transverse RV strain was an independent predictor of survival in this group of patients . In patiIn patients with different cardiomyopathies, connective tissue disease, and coronary artery disease, RV involvement is frequently seen. Conventional parameters of RV systolic function could not always detect subtle and subclinical RV changes and therefore RV longitudinal strain might help not only in detection but also in stratification of risk of patients with these cardiovascular conditions to develop the adverse event. RV longitudinal strain  emerged as an independent predictor of adverse events in addition to clinical parameters and LV function and therefore should be routinely evaluated during echocardiographic examination in these patients.


A review of the available literature was performed in order to summarize the existing evidence between osteoblast dysfunction and clinical features in non-hereditary sclerosing bone diseases. It has been known that proliferation and migration of osteoblasts are concerted by soluble factors such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), bone morphogenetic protein (BMP) but also by signal transduction cascades such as Wnt signaling pathway. Protein kinases play also a leading role in triggering the activation of osteoblasts in this group of diseases. Post-zygotic changes in mitogen-activated protein kinase (MAPK) have been shown to be associated with sporadic cases of Melorheostosis. Serum levels of FGF and PDGF have been shown to be increased in myelofibrosis, although studies focusing on Sphingosine-1-phosphate receptor was shown to be strongly expressed in Paget disease of the bone, which may partially explain the osteoblastic hyperactivity during this condition. Pathophysiological mechanisms of osteoblasts in osteoblastic metastases have been studied much more thoroughly than in rare sclerosing syndromes: striking cellular mechanisms such as osteomimicry or complex intercellular signaling alterations have been described. Further research is needed to describe pathological mechanisms by which rare sclerosing non hereditary diseases lead to osteoblast dysfunction. Sclerosing bone diseases are a heterogeneous group of skeletal alterations that share the process of impaired bone ossification. They are mostly rare diseases that are usually classified in hereditary and non-hereditary bone diseases. Hereditary sclerosing bone diseases are caused by genetic alterations resulting in increased bone formation and are represented by osteopetrosis, osteopoikilosis, pyknodysostosis, progressive diaphyseal dysplasia, osteopathia striata, hereditary multiple diaphyseal sclerosis andhyperostosis corticalis generalisata. Non-hereditary sclerosing bone diseases include intramedullary osteosclerosis, melorheostosis, Paget disease of bone, Erdheim-Chester disease, myelofibrosis and sickle cell disease. The diagnostic process of these conditions is often challenging. The resulting osteocondensation seen in imaging may be the final stage of an impaired process of stem cells differentiation which is in turn the consequence of altered levels of soluble growth factors. The basic laboratory tests in the diagnostic process of osteocondensation should include blood count, erythrocyte sedimentation rate, protid electrophoresis, transaminases, serum creatinine, calcium, bone alkaline phosphatases (bALP) and prostate specific antigen (PSA). Histology may also be required in order to exclude the diagnosis of hematological neoplasms. Impaired levels of soluble factors influencing osteoblast (Ob) metabolism such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) or bone morphogenetic protein (BMP) or alterations of intracellular pathways such as Wnt signaling, Mitogen-activated protein kinase (MAPK) signaling, Receptor activator of NF-κB (RANK)/ RANK ligand (RANKL)/ Osteoprotegerin (OPG) pathway have been described in this group of diseases. Ob metastases showed peculiar mechanisms inducing osteosclerosis such as cross-talk between cancer cells and Ob. The aim of this review is to describe the main clinical features in non-hereditary sclerosing bone diseases and to review the existing evidence concerning the Ob dysfunction involved in the physiopathological mechanism of these disorders.Osteoblasts (Ob) originate from mesenchymal cells, secrete matrix proteins and promote mineralization during the bone modelling and restructuring process . Ob are p.S264 substitutions increase transforming growth factor β (TGF-β) signaling, which in turn leads to an increased Ob proliferation and a consequent bone sclerosis [Melorheostosis is a rare mesenchymal dysplasia consisting of a local massive sclerosis of the bone. Clinical findings of this disease depend on the location of the bone involved and include local pain, stiffness, limited range of motion, limb swelling. Long bones of the upper or lower extremities are usually involved, while axial skeleton is rarely affected . Typicalclerosis ,18. Postclerosis  (Table 1Intramedullary osteosclerosis (IMOS) is an uncommon condition not associated with family history, systemic disease, or triggers. IMOS shows adult onset, female predominance and local pain; nevertheless, no specific signs and symptoms characterize this disease and the diagnosis can only be made after excluding other diseases. The lower limbs are usually involved and tibial diaphysis is the most commonly involved site . The radProstate cancer (PCa) is the prototype of cancer with a predilection for generating osteoblastic metastases. The disordered bone architecture of bone metastases defines a structural weakness prone to fracture . Since pAlong with essential thrombocytopenia and polycythemia vera, idiopathic primary myelofibrosis (IMF) is a myeloproliferative neoplasm, a chronic disease of the bone marrow. Clinical manifestations in IMF include severe anemia, hepatosplenomegaly, bone pain, fatigue, night sweats, fever, cachexia and splenic infarction. Its prognosis is unfavorable, burdened by leukemic progression that occurs in 20% of patients, but also by the consequences of cytopenias such as infections or bleedings and the average survival is about 5 to 7 years. Bone marrow morphology is critical for a correct diagnosis showing bone marrow sclerosis associated with JAK2, CALR or MPL mutations observed in 90% of patients . In IMF,p.Glu6Val in HBB (Hemoglobin subunit Beta) and a second HBB pathogenic variant which results in the anomalous hemoglobin polymerization, thereby causing multiorgan infarctions. The most common type is known as sickle cell anemia. SCD encompasses several signs and symptoms such as intermittent vaso-occlusive events, chronic hemolytic anemia; bone involvement is the most common clinical manifestation [Sickle cell disease (SCD) is a group of blood inherited disorders characterized by the presence of at least one hemoglobin S allele (HbS) estation , which cestation  assessedestation  demonstrestation . Hydroxyestation . Novel testation .Osteoarthritis (OA) is a joint chronic disease, which causes damage to the cartilage and surrounding tissues. The pathogenesis of cartilage degradation is strictly related to concomitant changes in subchondral and periarticular bone, characterized by joint surface eburnation, subchondral irregular thickness, sclerosis, and osteophyte formation .The main clinical symptoms are pain, stiffness and loss of function; radiological findings include joint space narrowing, geodes and subchondral bone sclerosis. Although risk factors such as high BMI, female gender, muscle weakness, genetic predisposition, prior joint injury are clearly associated with the development of OA , the exaIt is known that an inflammatory process is associated with the alteration of the joint cartilage. OA is characterized by several stages; in the advanced stages, sclerosis of the subchondral bone is observed while in the early stages the subchondral bone thins ,71. In tBRAFV600E, shown in 57-75% of cases [BRAF-V600-mutant ECD Patients with severe heart or neurological involvement, BRAF-inhibitors such as vemurafenib or dabrafenib are recommended [Erdheim-Chester Disease (ECD) is a rare form of non-Langerhans cell histiocytosis that was first reported by Chester and Erdheim in 1930 . ECD usuof cases . This muommended ; if thisommended .Paget disease of the bone (PDB) is a metabolic bone disorder characterized by an unbalanced turnover of bone tissue affecting one or more bones. Bone pain is the most common symptom and typically worsens at rest or during the night. In a series of cases, deafness was evaluated in 7.9% of the patients . EntrapmFocal bone changes are characterized by hypertrophic and giant osteoclast clusters containing up to 100 nuclei, while normal osteoclasts show up to 20 nuclei . Ob hypeThe pathogenic mechanisms underlying non hereditary sclerosing bone diseases are not yet fully understood. Several diseases in this group such as osteoarthritis or osteoblastic metastases have been better characterized thanks to more in-depth research, while the rare sclerosing bone syndromes need further research in order to enlighten the underlying alterations. Several intracellular proteins and specific gene expression in Ob have been shown to play a major role in the development of these syndromes. In melorheostosis, SMAD3 mutations may be the main factor in causing increased proliferation of Ob. Activation of the MAPK pathway may play a major role in the genesis of sclerotic lesions. Upregulation of MAPK was observed in both melorhestosis and ECD; in this hystiocytosis the BRAFV600E mutation is associated with a constitutive activation of MAPK signal. Wnt proteins play a leading role in regulating osteocyte proliferation and differentiation; dysfunctions of these proteins have been observed in the genesis of sclerosing lesions in osteoblastic metastases and osteoarthritis, where upregulation of PO defines the activation of wnt/beta-catenin signaling. Ob hyperactivity in PDB may result from increased expression of S1P on the surface of osteoclasts. In sickle cell anemia, iron overload has been shown to hamper osteoblastic activity. High serum levels of FGF, PDGF and TGFβ could drive the sclerosing process in bone marrow of patients with IMF, while OPG level was decreased. Further research is needed to understand the etiopathogenetic mechanisms of non-hereditary sclerosing bone diseases, as well as to develop targeted therapies to reduce bone complications and improve the outcomes of this set of diseases.