This paper constructs planar-type graphene thin film current collectors for proton exchange membrane fuel cells (PEMFCs). The present planar-type current collector adopts FR-4 as the substrate and coats a copper thin film using thermal evaporation for the electric-conduction layer. A graphene thin film is then coated onto the current collector to prevent corrosion due to electrochemical reactions. Three different coating techniques are conducted and compared: Spin coating, RF magnetron sputtering, and screen printing. The corrosion rates and surface resistances are tested and compared for the different coating techniques. Single cell PEMFCs with the developed current collectors are assembled and tested. A PEMFC module with two cells is also designed and constructed. The cell performances are measured to investigate the device feasibility. The proton exchange membrane fuel cell (PEFMC) adopts hydrogen as its fuel, and the fuel energy is converted into electrical energy through an electrochemical reaction. In portable applications, bipolar plates or current collectors account for the major weight percentage among PEMFC components . In portIn order to shrink the fuel cell size for portable applications, Lee et al. first proposed the micro-electro-mechanical system (MEMS) technique to construct a micro fuel cell . After tAlthough good electrical conductivity is important for micro fuel cell current collectors, corrosion resistance is also very important, as poor corrosion resistance degrades micro fuel cell durability. Related research has been conducted on current collector electrical conductivity, such as coating gold or TiN/TiAlN layers onto stainless steel expanded meshes , depositThe printed-circuit board (PCB) technique was also applied to construct the planar-type portable fuel cell, which was first proposed by O’Hare et al.  and SchmTo avoid distortion due to the mismatch of significantly different material properties between the metal current collector and FR-4 substrate in the PCB fuel cell, a lightweight current collector was proposed by Sung et al.  and KuanA single cell PEMFC was also assembled and tested . Ning etIn addition, the precious metal coating of the current collector is essential for improving corrosion resistance; however, it would increase the total cost of the cell. Optimizing the coating thickness is essential to maintain the balance between the corrosion resistance and cost. Surfaces and interfaces present a significant portion of the workable area and a network of energetically mismatched or metastable molecules, which could be exploited to either control surface reactions, engineer bulk stability, or reveal new fundamental details of those now well understood processes or systems . Lee et The main objective of this paper is to develop lightweight planar-type current collectors for a PEMFC module with two cells. The lightweight current collector also adopts FR-4 as the substrate material; the copper thin film is coated via the thermal evaporation process that functions as the electric-conduction layer. A graphene thin film is further coated onto the copper thin film as the corrosion-resistance layer. The proposed planar-type current collectors were fabricated with three different graphene thin film coating processes. The completed current collectors were assembled into a two-cell PEMFC module. Related performance and stability experiments were also performed.The present lightweight current collector for the two-cell PEMFC module consists of three layers. The first layer is the FR-4 substrate. The second layer is the electric-conduction layer–copper thin film fabricated by adopting the thermal evaporation process to coat copper particles onto the FR-4 surface. The third layer is the corrosion-resistance layer–graphene thin film coating via three different processes discussed and compared in this paper.A geometric drawing of the two-cell current collector is shown in The fabrication process for the electrical conduction layer is shown in The fabrication process for the graphene thin film using graphene ink is shown in The graphene thin film fabrication process uses a graphene suspension, as shown in The graphene thin film fabrication process using graphene dispersion is shown in The measurements on the weight and thickness of the current collectors are shown in The four probe resistance measurements were conducted to ensure electrical conductibility uniformity for the complete current collectors. An illustration of the five current collector monitoring points for surface resistance measurements is shown in The current collector surface resistance values with a graphene thin film using graphene ink, graphene suspension, or graphene dispersion, for the complete current collectors, are shown in Preventing corrosion is also very important for the current collectors. Therefore, the Tafel extrapolation method is conducted to measure the corrosion characteristics. An aqueous methanol solution and de-ionized water are first prepared and mixed as an acidic aqueous methanol solution, the mix ratio of methanol to de-ionized water is 1:9 and the PH value is 6.2. A sample of the fabricated current collector would then be placed in the solution. The Ag/AgCl (3.5M KCl) is adopted as the reference electrode with a platinum counter electrode. Impedance measurements were conducted adopting the BioLogic SP-150AC instrument. The scan rate is 0.166 mV/s and the voltage is −0.025–0.025 V. The results of the Tafel curves are shown in Scanning electron microscopy (SEM) is adopted to investigate the micrographs of the cross-sections of the current collectors coated with different graphene thin films, and this is shown in −2 at the anode, and Pt 0.4 mg cm−2 at the cathode. Silicon gaskets with a 0.15 mm thickness were used for sealing. The cell compression ratio was 34.78%.After completed current collector fabrication, the proposed current collectors were assembled into a two-cell PEMFC module to measure cell performance. PEMFC module with both forced convection air-breathing cathodes and self-air-breathing cathodes were studied. The MEA adopted in this paper is a three-layer catalyst coated type with a DuPont Nafion HP membrane and carbon paper diffusion layers. The reaction area was 5 × 5 cm, the catalyst load was Pt 0.1 mg cmAn exploded drawing of the two-cell PEMFC module with a forced convection air-breathing cathode is shown in The PEMFC module performances with forced convection air-breathing and three different types of graphene coatings were conducted at different anode hydrogen and cathode air flowrates. The anode hydrogen flowrate and cathode air flow rate ratios were kept at 1:2 in the experiment. Both the anode hydrogen flow and cathode airflow were humidified and kept at 50 °C. The anode hydrogen/cathode air flow rates of 25/50, 50/100, 100/200, 200/400, 300/600, and 400/800 sccm were investigated.A performance comparison of the PEMFC module with a forced air-breathing cathode and the corrosion-resistance layer using graphene ink coating is shown in −2 at the low flow rate (25/50 sccm), the further reduction of the operating voltage leads to a sharp drop of the current. This is due to the insufficient reactant, and the fuel cell becomes highly unstable, the cell performance is sharply dropped and is without a smooth transition through the ohmic loss to the concentration loss. Therefore, the low flow rate condition is not suitable for the cell operation at the high-current range for the current collector with the graphene ink thin film.In the high-current range of higher than 75 mA cmThe PEMFC module performance is decreased when the flow rates are increased. The PEMFC module performance comparison with a forced air-breathing cathode and a corrosion-resistance layer using a graphene-suspension coating is shown in −2 at the low flow rate (25/50 sccm), the further reduction of the operating voltage will lead to a sharp drop of the current. The results also show a similar trend to the current collectors with a graphene ink coating and the related explanation refers to the The performance comparison of the PEMFC module with a forced air-breathing cathode and the corrosion-resistance layer using a graphene-dispersion coating is shown in The results show that the current collectors with three different types of graphene thin film show similar phenomena and trends. In addition, the current collector using the graphene-dispersion coating presents the highest performance, using graphene suspension is second, and using graphene ink presents the lowest PEMFC module performance.The PEMFC module performances with self-breathing and three different types of graphene coatings were further conducted at the anode hydrogen flow rate of 100 sccm. The anode hydrogen flow was humidified, kept at 50 °C and a 100 sccm flow rate. The cathode side was open for self-air-breathing and the ambient temperature was 25 °C. The performance comparison of the PEMFC module with self-air-breathing cathode and the corrosion-resistance layer is shown in Although the surface resistances are roughly the same for all cells via different graphene thin film coatings in According to the measurements in After investigating the PEMFC module performance with the developed current collectors for both forced air-breathing and self-air-breathing cathodes, long-term stability tests of the PEMFC modules, with self-air-breathing cathodes, using three different graphene coatings as corrosion-resistance layers, were further conducted. Experiments were performed at a 100 sccm anode hydrogen flow rate and the hydrogen was humidified and kept at an inlet fuel temperature of 50 °C. The cathode side is open for self-air-breathing and the ambient temperature is 25 °C. The PEMFC module stability tests with self-air-breathing cathodes and different types of corrosion-resistance layers under 1 V loads, are shown in The results show that both the PEMFC modules with current collectors using graphene-dispersion and graphene-suspension coatings are quite stable. The PEMFC module performance with the collectors using the graphene-dispersion coating is slightly higher than that using the graphene suspension. Although the PEMFC module with current collectors using the graphene ink coating also showed good stability, the PEMFC module performance is distinctly lower than those using the graphene dispersion and graphene suspension. The results also successfully show the feasibility of the developed current collectors in portable applications. However, the tests only represent approximately a 10 h operation period, which is much less than the anticipated life specification. Therefore, additional longer duration tests, particularly under realistic cycling conditions that accelerate degradation, should be made in the future for realistic commercialized applications .This paper proposes lightweight current collectors and constructed a PEMFC module with two cells. The current collectors consist of a FR-4 substrate, a copper thin film as the electric-conduction layer, and a graphene thin film as the corrosion-resistance layer. The copper thin film is accomplished by a thermal evaporation process. Three different graphene coating processes to fabricate the graphene thin film were investigated. The results show that adopting a graphene-dispersion coating has the best corrosion-resistance capability, the graphene-suspension coating is second, and graphene ink is the lowest. Further study of the fabricated current collectors assembled into a two-cell PEMFC module show that the current collectors with a graphene-dispersion coating have the highest PEMFC module performance, the graphene-suspension coating is second, and graphene ink also has the lowest PEMFC module performance. The graphene-dispersion thin film shows the lowest coating weight and superior surface flatness, and the graphene ink thin film shows a significantly higher coating weight, voids inside, and an uneven surface in the testing results. Long-time stability studies were also conducted, and all three graphene coating processes showed good PEMFC module stability after long-term operations. During the long-time stability tests, the PEMFC module performances for the current collectors that used a graphene-dispersion coating are a little bit higher than for the current collectors that used a graphene-suspension coating. These performances were both clearly higher than those of current collectors that used a graphene ink coating. The feasibility studies also showed the potential of the developed current collectors in portable applications.


Disasters are associated with loss of property and loss of psychological well-being. Receiving various types of social support, such as emotional or tangible support, from various sources, such as family or friends, have been found to reduce the adverse impact of a disaster on affected adult community residents. It is not well studied, though, why some adults will not seek or provide support after a disaster while other adults will. The purpose of this study was to explore how age might play a role in how comfortable adults were in seeking social support from others and how age might play a role in how comfortable adults were in providing social support to others after a disaster, the 2014 San Diego County, CA wildfires. One hundred and twenty-two community residents (18 to 80 years) were recruited with 33 adults identified as Secondary Disaster Survivors and 89 adults identified as Non-Victims. Analyses revealed that age did play a role in Secondary Survivors’ comfortableness in seeking support from others; Older Adults (50-80) were significantly less likely than Middle-aged Adults (30-49) to feel comfortable in seeking support. There was no significant comfortableness difference between Middle-aged and Younger Adults (18-29) in seeking support. There were no age group differences in comfortableness in providing support to others for Secondary Survivors and Non-Victims. Implications from this data suggest that local disaster organizations should actively support affected older adults post-disaster while also including unaffected adults of all ages in the process of providing support to those in need.


Strategies for delivering nucleic acids into damaged and diseased tissues have been divided into two major areas: viral and non-viral gene therapy. In this mini-review article we discuss the application of gene therapy for the treatment of osteoarthritis (OA), one of the most common forms of arthritis. We focus primarily on non-viral gene therapy and cell therapy. We briefly discuss the advantages and disadvantages of viral and non-viral gene therapy and review the nucleic acid transfer systems that have been used for gene delivery into articular chondrocytes in cartilage from the synovial joint. Although viral gene delivery has been more popular due to its reported efficiency, significant effort has gone into enhancing the transfection efficiency of non-viral delivery, making non-viral approaches promising tools for further application in basic, translational and clinical studies on OA. Non-viral gene delivery technologies have the potential to transform the future development of disease-modifying therapeutics for OA and related osteoarticular disorders. However, further research is needed to optimize transfection efficiency, longevity and duration of gene expression. Osteoarthritis (OA) is a leading cause of disability across the world and a major cost contributor to health and social care systems , using xenografts, or biomaterial implants as well as partial or total joint replacement surgery are the most effective and current surgical interventions to improve quality of life in OA patients   or medicinal signaling cells, as proposed by Arnold Caplan , whose gene was transduced using a retroviral vector into this protein packaging cell line , interleukin 1β (IL-1β), and nerve growth factor (NGF), was intra-articularly injected in a surgically induced OA in mice , polyethylenimine (PEI) or polyethylene glycol (PEG) are also of great interest for gene delivery into cells. These polymers form strong electrostatic complexes with nucleic acids, which facilitates their entry into cells , promoting ECM production in vivo, as compared to control group , which interacts specifically with chondrocytes was covalently modified to bind to CAP forming a non-viral vector -conjugating systems as a disease-modifying strategy for OA. Nano-micelles of PEG carrying the cartilage anabolic factor runx-related transcription factor-1 (RUNX1) mRNA have been shown to significantly suppress the progression of OA after intra-articular injection in a mouse OA model  and has received research funding from Kolon TissueGene and Merck KGaA, Darmstadt, Germany. The remaining 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.


Preclinical animal models of breast cancer provide the opportunity to identify chemopreventive drugs with single-agent activity as well as effective multi-modality regimens for primary as well as secondary prevention in high-risk persons. Our group has used the 7,12-dimethylbenz(a)anthracene (DMBA) mouse model of carcinogen-induced breast cancer to explore the clinical potential of two tyrosine kinase inhibitors and a nucleoside analog as chemopreventive agents. All three agents exhibited promising preclinical activity both as monotherapy and as components of combination therapy with the standard chemotherapy drug paclitaxel. The tumors developing despite chemoprevention were not only small and grew slowly, but they also displayed a uniquely more pro-apoptotic protein expression profile. Hence, our experimental chemopreventive drugs were capable of preventing the development of aggressive mammary gland tumors with an apoptosis-resistant protein expression profile. Guidelines have been developed by the US Preventive Services Task Force (USPSTF)  and the American Society of Clinical Oncology (ASCO)[10] regarding the rationale and data-driven, evidence-based use of endocrine therapy for women at high risk for breast cancer. Precision medicines, as well as targeted biotherapeutic agents such as Trastuzumab (Herceptin), offer the opportunity for patient-tailored secondary prevention of breast cancer . The discovery of additional effective chemopreventive strategies using chemotherapy drugs, precision medicines, biologics, and natural compounds is a major area of translational research emphasis in contemporary oncology [13]. Over 1.5 million women are diagnosed with and over 0.5 million women die of breast cancer each year. Although breast cancer mortality has declined over the past two decades, breast cancer remains the leading cause of cancer deaths in women aged 20 to 59 years . The adverse impact of breast cancer on quality of life, productivity, and survival, as well as health care costs, have prompted intensive research efforts aimed at the identification of breast cancer prevention methods [3]. Effective chemoprevention strategies with selective estrogen receptor modulators (SERMs), such as tamoxifen, and aromatase inhibitors (AIs), including steroidal inhibitors such as exemestane, and nonsteroidal inhibitors, such as anastrozole and letrozole, have been developed to decrease the risk of both invasive and noninvasive breast cancer . An analysis of 83.399 women at high risk for breast cancer who received SERMs for primary chemoprevention showed a 38% reduction in breast cancer incidence [5]. AIs administered as adjuvant therapy after breast surgery were highly effective in reducing the incidence of breast cancer recurrence in women who are diagnosed with breast cancer [6]. In a randomized exemestane study designed to detect a 65% relative decline in invasive breast cancer, 4560 eligible postmenopausal women aged 35 or over had at least one of the following risk factors:in vivoanimal model for carcinogen-induced breast cancer. The purpose of the current review is to summarize the salient features of our studies as well as discuss the insights and lessons learned from biomarker analyses. Our recent drug discovery efforts have focused on a data-driven strategy to identify active chemopreventive agents for breast cancer by leveraging anMice treated with the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) develop mammary gland tumors that morphologically resemble human myoepithelial carcinomas and myoepitheliomas [14]. Adenosquamous carcinomas and ductal carcinomas have also been reported in DMBA-treated mice [15].The polycyclic aromatic hydrocarbons like DMBA activate the aryl hydrocarbon receptor (AhR), which activates a signal transduction cascade that leads to the formation of the mutagenic epoxide of DMBA via cytochromes P450 enzymes. DNA damage due to this mutagenic epoxide is the proposed driving mechanism of malignant transformation in breast tissue [16]. The DMBA initiates and affects multiple steps of carcinogenesis, starting from the inhibition of differentiation in the early stages and followed by cell cycle disruptions such as upregulation of Cyclin D1 and c-Myc, possibly via nuclear factor-κB and Wnt pathways [17]. Disruptions of intracellular members of cell cycle regulation such as PI3K/mTOR/AKT and PTEN were also seen later in the disease course similar to luminal type human breast cancers [18]. In a series of studies, we have used the DMBA model of breast cancer to evaluate the chemopreventive activity of promising new agents in side by side comparison with the standard antibreast cancer drug paclitaxel. All control mice treated with DMBA developed mammary gland tumors within 16 weeks. The median time for progression-free survival (in other terms time to tumor development) was 13 weeks for these mice. The standard breast cancer drug paclitaxel preventedor slowed down the development of mammary tumors significantly and more than 25% of rodents remained tumor-free even after 23 weeks. The progression-free survival was increased to 16 weeks in the paclitaxel arm also .In preclinical models, the phosphoramide derivatives of nucleoside analogs showed anti-tumor activities and emerged as potential anticancer drugs [19]. Stampidine is among these agents and it’s an aryl phosphate derivative of stavudine. Its clinical activity was first tested as an anti-HIV agent in preclinical models and also in a phase 1 study[20]. The drug was well-tolerated without dose-limiting toxicities [20]. Stampidine has been shown to act as an epigenetic regulator of gene expression by methylating and silencing the several oncogenic transcription factors[21]. We tested the chemopreventive activity of stampidine in the DMBA model. When co-administered with DMBA, stampidine decreased the number and size of mammary gland tumors that have developed after the DMBA challenge. In addition, the combination of stampidine and paclitaxel synergistically reduced the tumor burden. Another hypothesis-generating point from our study was the demonstration of higher proapoptotic and lower antiapoptotic protein expression in tumors developing in stampidine-treated mice [22]. These results demonstrated that stampidine prevents the development of aggressive breast tumors DMBA challenge. The chemopreventive potency of stampidine was similar to that of paclitaxel as well as two potent kinase inhibitors, WHI-P131  [22–25] and LFM-A13 (an inhibitor of TEC and PLK family kinases) [26] . These findings illustrate the potential of stampidine as a chemopreventive agent against breast cancer. In regards to the two lead kinase inhibitors we tested, WHI-P131 was active in the DMBA induced cancer model. The median tumor free-survival was 18 weeks with WHI-P131 with 20% of mice were tumor in the 18th week of DMBA treatment. This tumor-free survival was significantly improved compared to the control arm (P £ 0.001). The antitumor efficacy was similar to that of paclitaxel (P =0.544)  [25]. We think that the combination of WHI-P131 with conventional chemotherapeutics can further improve the antitumor efficacy of WHI-P131 as a chemopreventive agent.The other novel agent we evaluated was LFM-A13 (19). In the DMBA model, LFM-A13 significantly increased the survival to 40% compared to 15% in the control arm. The combination of paclitaxel and LFM-A13 further increased the survival rate to 50% in the 25th week. The tumor incidence and also tumor burden were significantly reduced in the LFM-A13 group [26]. LFM-A13 prolonged the tumor-free interval by 2 weeks  compared to the control arm. 30% of mice treated with LFM-A13 were tumor-free at 20th week demonstrating long-lasting chemoprevention with the agent. The antitumor efficacy was similar to that of paclitaxel and WHI-P131 .Notably, the tumors developing despite chemoprevention with the kinase inhibitors or stampidine were not only small and grew slowly, but they also displayed a uniquely more proapoptotic protein expression profile. Hence, our experimental chemoprentive drugs are capable of preventing the development of aggressive mammary gland tumors with an apoptosis-resistant protein expression profile. Although the use of tamoxifen and AI’s for secondary prevention have significantly reduced the risk of are recurrence in hormone receptor-positive breast cancer after standard first-line therapy , many high-risk patient populations  experience a recurrence. Optimizing secondary prevention with drugs used in the adjuvant and prolonged maintenance therapy settings may contribute to a clinically meaningful improvement of survival outcome. Our data in the DMBA model suggest that stampidine, WHI-P131, and LFM-A13 may be useful as chemopreventive agents and as components for secondary chemoprevention protocols. In the DMBA model, the efficacy of these agents in chemoprevention was similar to that of paclitaxel which is among the most effective breast cancer therapies [27]. We are planning to evaluate their activity in combination with tamoxifen and AIs for secondary prevention considering the potential to inhibit different steps of oncogenesis due to different mechanisms of antitumor action. The enhanced potency observed when these agents were combined with paclitaxel is also deserving of further study . LFM-A13 and WHI-P131 were also tested for activity in MMTV/Neu transgenic HER-2 positive models of breast cancer , so their synergism with trastuzumab is another area that warrants further research.As mentioned earlier, the tumors developing despite chemoprevention with the kinase inhibitors or stampidine displayed a pro-apoptotic protein expression profile, which was different from the protein expression profile of aggressive tumors developing in DMBA-treated control mice [22]. Hence, our experimental chemopreventive drugs are capable of preventing the development of aggressive mammary gland tumors with an apoptosis-resistant protein expression profile. It is our working hypothesis that recurrent tumors emerging after chemoprevention with these chemopreventive drugs will, therefore, be substantially more sensitive to available salvage chemotherapy regimens in standard second-line settings. This should contribute to an improved treatment and survival outcome after recurrence. One of the most important properties of these rationally designed agents is their tolerability which increases their potential to be used in the combinations. All three agents were well tolerated without dose-limiting hematologic, renal and hepatic toxicities in multiple animal studies . Stampidine did not cause any dose-limiting toxicities in a phase 1 study of thirty HIV patients [20]. However, LFM-A13 and WHI-P131have not been evaluated in human subjects and all three drugs remain to be evaluated to breast cancer patients. In summary, stampidine, LFM-A13, and WHI-P131 showed potent chemopreventive activity by reducing the development and number of breast tumors and improving the tumor-free survival outcome in breast cancer animal models. Further models and studies testing the synergism of these agents with another chemopreventive agent like tamoxifen and AI’s and also the use of these agents for primary chemoprevention in women at high risk for breast cancer would seem warranted.


However, it is becoming increasingly evident that the “cytokine storm” induced by aberrant activation of the innate immune response against a viral pathogen may sometimes offer replicative advantage to the virus thus promoting disease pathogenesis. Thus, it is important to fine tune the responses of the innate immune network that can be achieved via a deeper insight into the candidate molecules involved. Several pattern recognition receptors (PRRs) like the Toll like receptors (TLRs), NOD-like receptors (NLRs), and the retinoic acid inducible gene-I (RIG-I) like receptors (RLRs) recognize cytosolic RNA viruses and mount an antiviral immune response. RLRs recognize invasive viral RNA produced during infection and mediate the induction of Type I Interferons via the mitochondrial antiviral signaling (MAVS) molecule. It is an intriguing fact that the mitochondrion, one of the cell’s most vital organelle, has evolved to be a central hub in this antiviral defense. However, cytokine responses and interferon signaling via MAVS signalosome at the mitochondria must be tightly regulated to prevent overactivation of the immune responses. This review focuses on our current understanding of the innate immune sensing of the host mitochondria by the RLR-MAVS signalosome and its specificity against some of the emerging/re-emerging RNA viruses like Ebola, Zika, Influenza A virus (IAV), and severe acute respiratory syndrome-coronavirus (SARS-CoV) that may expand our understanding for novel pharmaceutical development.As the world faces the challenge of the COVID-19 pandemic, it has become an urgent need of the hour to understand how our immune system sense and respond to RNA viruses that are often life-threatening. While most vaccine strategies for these viruses are developed around a programmed antibody response, relatively less attention is paid to our innate immune defenses that can determine the outcome of a viral infection  Evolutionary conserved signaling intermediate in Toll (ECSIT) pathway, a component of the mitochondrial complex I, has been shown to enhance TLR7 responses via the mitochondrial adaptor protein tumor necrosis factor receptor (TNFR) associated factor 6  belonging to Filoviridae family, Zika virus (ZIKV) belonging to Flaviviridae family, Influenza A virus (IAV) belonging to Orthomyxoviridae family, and severe acute respiratory syndrome-coronavirus (SARS-CoV) belonging to Coronaviridae family. These viruses have been known to cause deadly outbreaks across the world and it is important to analyze whether key sensors of RNA viruses like the RIG-I/MAVS pathway are important targets of these viruses either to suppress of hyper-activate the immune responses.via CARD-CARD interaction  and a DECH-helicase domain required for RNA binding and ATP hydrolysis . All theeraction . MAVS is cascade . Followi cascade . This st cascade . Interfevia MAVS . Over cevia MAVS . WhetherFollowing viral infection, our cellular defense machinery systematically induces a number of cytokines (both pro‐ and anti-inflammatory) that, in certain instances, may lead to hyperstimulation of the immune response in a positive feedback loop . This levia mitochondrial sensing  is an emerging pathogen and has an almost 90% mortality rate. Although it is mostly endemic to West Africa with the Democratic Republic of Congo (DRC) being the hardest hit region over the past decade (2013–2019), it remains a major health concern worldwide due to its high potential to infect other species and the unavailability of a viable therapy till date . EBOV isvia binding to transiently produced dsRNA during EBOV infection, thus preventing viral sensing and also by binding to the RIG-I ATPase activator PACT  infection demonstrated that IFN-dependent and independent MAVS signaling takes place in an organ specific manner, where activation of monocytes and subsequent trafficking to the spleen occurs in a MAVS-dependent manner . EBOV mitor PACT . In an ilocation . FurtherFlavivirus. However, apart from mosquito-transmission, several other modes of ZIKV infection have been reported and the most striking is the mother to fetus transmission via the transplacental route (Zika virus (ZIKV) is a re-emerging mosquito borne pathogen belonging to the genus al route . Althougal route . The re-via K48 linked polyubiquitination and subsequent downregulation of IFNβ pathway  single stranded RNA virus that has shown to co-evolve with the host and strongly antagonizes the host antiviral IFN-responses. Several non-structural proteins of ZIKV like NS1, NS2B/3, NS4A, NS4B, and NS5 have been shown to antagonize IFN-responses . Studies pathway . Further pathway . Studies pathway . HoweverInfluenza A virus (IAV) is one of the four types of influenza virus and the only influenza virus sub-type that has been known to cause global pandemic. Based on the presence of two surface proteins, hemagglutinin (HA) and neuraminidase (N), IAV can be sub-categorized into different strains . The 191in vitro (via its interaction with MAVS/TRIM2/Riplet (via its direct interaction with the RIG-I/MAVS pathway (The Influenza virus contains eight segmented (−) single stranded RNA and affects the upper respiratory tract epithelial cells causing the “seasonal” flu or fatal pulmonary disorder in extreme conditions . The polin vitro . Besidesin vitro . Inflamm2/Riplet . TRIM25 2/Riplet . This co2/Riplet . However pathway . Thus, NCoronavirus is emerging pathogens that has serious life-threatening impact on human health. Severe acute respiratory syndrome-related coronavirus (SARS-CoV1) caused a major outbreak of respiratory disease in 2002–2004 . The curRNA viruses have become important etiological agents of emerging pathogens in humans constituting a major percentage of all human emerging diseases including those induced by bacteria or parasites. The past decade has seen several cases of pandemics arising due to RNA viruses originating from wild life reservoirs like the Ebola, H1N1 influenza, SARS, and MERS and the recent COVID-19 pandemic. The RNA polymerases of these viruses often lack proofreading activity increasing their mutation rates during the replicative stage of the virus. This comes as a severe challenge in developing vaccine strategies and it is important to understand conserved host immune responses which may help combat a wide range of these RNA viruses.via the production of Type I Interferon, is often targeted by the viruses for the successful establishment of an infection. However, priming of IFN-responses prior to an infection can be a double edged sword as cytokine storm following hyper-stimulation of the immune responses and the over production of pro-inflammatory cytokines have been shown to be associated with diseases like Ebola, Influenza, and COVID-19 (The innate immune response, which provides the first line of defense against these RNA viruses COVID-19  and the PM conceived the work. SD, ND, and PM wrote the manuscript. PM prepared the figures and revised the entire manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The insulin-like growth factor (IGF) system regulates metabolic and mitogenic signaling through an intricate network of related receptors and hormones. IGF-II is one of several hormones within this system that primarily regulates mitogenic functions and is especially important during fetal growth and development. IGF-II is also found to be overexpressed in several cancer types, promoting growth and survival. It is also unique in the IGF system as it acts through both IGF-1R and insulin receptor isoform A (IR-A). Despite this, IGF-II is the least investigated ligand of the IGF system. This review will explore recent developments in IGF-II research including a structure of IGF-II bound to IGF-1R determined using cryo-electron microscopy (cryoEM). Comparisons are made with the structures of insulin and IGF-I bound to their cognate receptors. Finally discussed are outstanding questions in the mechanism of action of IGF-II with the goal of developing antagonists of IGF action in cancer. The insulin-like growth factor (IGF) system controls metabolic and mitogenic responses in mammalian cells and importantly regulates embryonic growth and development as well as adult growth . The IGFIgf2 leads to a 60% reduction in weight at birth . B. B63]. BAn as yet unexplained observation is the limited correlation of the site-directed mutagenesis data for IGF-II site 2 residues  and theiFor both IGF-I:IGF-1R and insulin:IR complexes additional structures have been described that have led to a proposed transient interaction of the ligand with a different site on the receptor. This may represent the first site of contact for the ligand or an intermediate site to facilitate conformational change of the ligand and receptor  as observed in IGF-I (Glu9) and insulin bound to IR (HisB10). The remaining residues identified by mutagenesis as contacting the receptor may be involved in transient interactions with the receptors. The same transient interaction is expected with IGF-II binding; however, this is yet to be observed. A detailed understanding of how IGF-II engages with its receptors and confers downstream signaling activation is essential in developing drug therapies that target IGF action in cancer. The relatively minor role of IGF-II in adult cell function means that blocking this pathway as a cancer therapy may have little effect on healthy adult cells whilst slowing cancer cell growth. Currently, most approaches target IGF action by directly blocking binding to IGF-1R:IGF-1R antibodies inhibit ligand binding and stimulate receptor internalisation . Such in


Surveys are key means of obtaining policy-relevant information not available fromroutine sources. Bias arising from non-participation is typically handled byapplying weights derived from limited socio-demographic characteristics. Thisapproach neither captures nor adjusts for differences in health and relatedbehaviours between participants and non-participants within categories. Weaddressed non-participation bias in alcohol consumption estimates using novelmethodology applied to 2003 Scottish Health Survey responses record-linked toprospective administrative data. Differences were identified insocio-demographic characteristics, alcohol-related harm  and all-cause mortality between survey participants and, fromunlinked administrative sources, the contemporaneous general population ofScotland. These were used to infer the number of non-participants within eachsubgroup defined by socio-demographics and health outcomes. Syntheticobservations for non-participants were then generated, missing only alcoholconsumption. Weekly alcohol consumption values among synthetic non-participantswere multiply imputed under missing at random and missing not at randomassumptions. Relative to estimates adjusted using previously derived weights,the obtained mean weekly alcohol intake estimates were up to 59% higher amongmen and 16% higher among women, depending on the assumptions imposed. This workdemonstrates the universal value of multiple imputation-based methodologicaladvancement incorporating administrative health data over routine weightingprocedures. For instance, there is likely to be a group of harmful and dependentdrinkers who may be disinclined to participate.Population health and health behaviour estimates are commonly derived from surveydata to monitor trends and formulate and evaluate policies. However, bias may ariseif the survey samples are not representative of the target population.Non-representativeness is of some concern when measures of association such asrelative risk are being estimated6 are usually applied in an attempt to correct for such unit non-response . However, these weights typicallyrely on a limited range of socio-demographic variables7 and are based on the assumption that non-participants have equivalentbehaviours to participants in the same socio-demographic category which is unlikelyto be the case.Survey weights derived from inverse probability weighting8 which is viable if the assumption that data are missing at random  holds. Alanya et al. applied MI to make unit non-response adjustmentsand compared it to weighting.9 They found MI to compare favourably, though not consistently so. In anothercomparison with weighting, MI showed comparable performance in terms of bias butalso yielded substantially lower variance estimates.10 However, these papers made no allowance for the data being missing not atrandom (MNAR: the probability of missingness is related to the unobserved data). Ifthe data are thought to be MNAR then an alternative approach is required, typicallyinvolving sensitivity analyses, and using methodology such as pattern mixture modelling11 among others.13An alternative to the application of survey weights is multiple imputation (MI),14 nations typically lack individual-level data amenable to forming the bases ofsampling frames. Thus, in countries such as the UK, individual non-participantscannot readily be identified and their routine health data extracted.Application of MI is strengthened if we can infer information on the absentnon-participants. In the absence of whole population registers, as existing inNordic countries,16 We consider both MAR and MNAR within a missing data framework,motivated by the possibility of non-participants differing in their alcohol consumption17 from survey participants with the same socio-demographic variables and healthoutcome statuses. Our approach involves: (1) exploitation of record-linkage tohospital discharges and mortality; (2) survey–population comparisons which informthe creation of synthetic partial observations for non-participants; and (3) MI togenerate refined estimates of weekly consumption of alcohol under assumptions of MAR and explorations of MNAR.18 We illustrate the application using data from the 2003 Scottish Health Survey(SHeS) individually record-linked to administrative health information from theScottish Morbidity Records (SMR), mortality data from the National Records ofScotland (NRS) and unlinked contemporaneous data for the entire population.We propose a novel methodology that aims to improve addressing non-participation biasin national health survey data in order to obtain less biased estimates of alcoholconsumption.In the next section, we provide the context and motivation for the methodologicalapproach described in section 3. In section 4, we report on the application beforediscussing the implications in section 5 and concluding in section 6.We aim to devise and apply methodology to estimate sex-specific adult populationmean alcohol consumption from national health survey data accounting for biasinduced by non-participation.19 Socio-demographic data available in the surveys include sex, age groupand Scottish Index of Multiple Deprivation , collectively referred tohere as ‘socio-demographic characteristics’. Alcohol consumption is calculatedin units  per week. Pre-derivedsurvey sampling weights which sum to the achieved sample total have been createdto account for the stratified, multi-stage random sample survey design anddepartures from population estimates by sex and age.19 We use the 2003 survey which had an adult response level of 60%.SHeS are a series of cross-sectional surveys designed to represent the Scottishpopulation living in private households.20) and mortality data using a probabilistic matching algorithm2124 (Baseline data on consenting SHeS participants (91%) have been confidentiallylinked to routinely-collected nationwide administrative health records availableuntil the end of 2011 providing prospective follow up of around eight years.These include prospective SMR which record hospital discharges  and the second indicating all-causemortality during follow-up. The analyses were restricted to individuals aged 20to 64 years in the survey year in an attempt to reduce the distortion ofinstitution-dwelling communities (e.g. older people living in care homes) –which are not in the sampling frame – on the survey-population comparisons.Our approach to addressing non-participation bias in alcohol consumption estimatesinvolved filling in the missing data in the survey in three stages marked as 1, 2and 3 in L denote the effective response level – thepercentage of the sample who both responded to the survey and consented tolinkage (determined by the product of the survey response level and the consentproportion and henceforth referred to as ‘response level’). We infer the totalsurvey size to be X1 is sex (1 formen and 2 for women), X2 is age group andX3 is area deprivation quintile. LetH1 corresponds to alcohol-related harm, andH2 corresponds to all-cause mortality. LetY denote the random variable for usual weekly alcoholconsumption as a semi-continuous measure (detailed in section 3.4.1).We use the following notation. Let S denote the binary random variable for the source of thedata, with R be the binary random variable for response, such thatw and have a mean of one.The observed linked survey data are therefore M be thetotal number of repeated independent data sets arising from MI.Let In stage 1, record linkage of survey data to SMR and NRS data was used todetermine the values of In stage 2, we made inference on the non-participants by comparing the nationalhealth survey data with corresponding population data to identify deviationsfrom representativeness in terms of R) as in the populationWe first assumed that This assumption is valid if the sampling frame for the survey is representativeof the general population.L (for which weighting may be ignored). Wehence identified the number of missing participants within eachsocio-demographic group in the survey as Y, set to missing, and w set to 1.Combining the participants with the synthetic observations for thenon-participants provides a data set for imputation.It follows that the M different bootstrap samples of sizeMdifferent imputed datasets. Theoretically, the bootstrap method could have beenextended to allow for uncertainty in Three modifications were needed to this general method. First, the method asproposed does not allow for uncertainty in the survey-based estimates ofSecond, the calculated numbers of missing participants in each category weregenerally not integers. To avoid possible bias due to rounding, we appliedrandom rounding which preserves the mean count. For example, if 2.6 missingparticipants were required in a particular category, then we took 3 missingparticipants with probability 0.6, and 2 missing participants with probability0.4. This was performed separately in each imputed data set.Third, estimates of n = 16) were imputed in the same wayas for non-participants.Once the synthetic observations for the non-participants were created at Stage 2,the unit (person) non-response problem had been converted into an item(variable) non-response problem with the synthetic non-participant observationshaving data on socio-demographic characteristics and health outcomes but missingdata on alcohol consumption. Imputation models for alcohol consumption couldthen be specified conditional on socio-demographic characteristics and healthoutcomes. Missing alcohol consumption observations among a small minority ofparticipants . We then accommodated the possibility of the databeing MNAR by allowing the distribution of alcohol consumption to differ in apre-specified manner between the non-participants and participants . Sections 3.4.1 and3.4.2 outline in turn the MI procedures based on MAR and MNAR.M data sets of synthetic non-participantobservations produced in stage 2, ultimately yielding Mmultiply imputed data sets. The imputed data sets were appended and weightedsubstantive analyses were performed using -mim-26 in Stata 13.1.For both MAR- and MNAR-based approaches, one stochastic imputation was performedfor each of the A2: Under MAR, conditional on socio-demographic characteristics and healthoutcomes, the distribution of alcohol consumption is independent ofparticipation status. This is assumption Y is a semi-continuous variable characterised by acombination of zeros representing those who do not report drinking and aright skewed continuous distribution of positive consumption. We followed apreviously adopted approach27 to handle the nontrivial proportion of zero values of alcoholconsumption by using a two-part model for Y, splitting itinto the dichotomous drinking status variable D andcontinuous consumption variable 28 with a shift parameter, k, selected to eliminate theskew of  = −1.9, 95% CI: −2.3 to −1.6). Predictive meanmatching with a potential match pool of size 10 was used to further improvethe imputations.Testing of the log transformation 29 within strata defined by sex and deprivation group to allow theassociation between harms and alcohol consumption to vary flexibly by sexand deprivation level. The w were entered as a continuouspredictor in both equations in equation . DifferD mayalso deviate from MAR . To assess thesensitivity of results to deviations from the MAR assumption forD, two categories of scenarios were considered for theimputation of D: first, imputing under a MAR assumption,and second, under an ‘upper bound’ scenario in which all non-participantsare set as drinkers for comparison. The rationale for this being of moresubstantive interest is that those who do not respond in any givensociodemographic and harm group are more rather than less likely to drink,and that it gives an interval which we can be certain about even if we lacka specific plausible deviation from MAR. We then modified the imputationprocedure for Our principal rationale for exploring MNAR concerns differential overalldrinking levels, but the possibility remains that H1 andby a modifying variable, in our case We consider the general specification which accommodates differentialmodification of the imputation model by X1; andsimilarly by X1.Various scenariosfor the magnitudes and signs of the Relative to participants with fully observed alcohol consumption, meanalcohol consumption is modified by Survey paradata-based approachWe considered two general approaches to specifying possible values forparameters 37 Here, invitedhouseholds who do not initially respond are re-approached one or more times,and the number of interviewer calls is recorded. Later respondingparticipants can be theorised to be increasingly more like non-participantswith the greater effort required to recruit them into the survey. We usedthe number of interviewer calls to a household as our proxy fornon-participation propensity, where an individual who responded in three(the median number) or fewer attempts is considered an early-participant,and those that took four or more attempts are considered late-participants.Estimates of  = 1.0 and  = 23.1 among men, and  = 0.75 and  = 1.17 among women, respectively.We drew on continuum-of-resistance theory which is predicated upon the ideaof a latent propensity to not participate.Literature-based approachWe also considered the scenario in which the consumption deviation from MARis twice the adjusted difference between early- and late-participants(MNAR2M and MNAR2UB). 38 For our purposes we posit this to be a generalisable estimate ofconsumption among drinkers who have been hospitalised. We thereforeconsidered the MNAR-based sensitivity analysis where the imputation modelinvolves specifying  = 309.7,  = 91.8; denoted MNAR5M and MNAR5UB). We alsoconsidered more moderate scenarios, where the deviation from MAR consumptionwas three-times the observed sex-specific mean among those whose experiencedharm  and finally wherethe deviation from MAR was equal to the observed sex-specific mean amongthose whose experience harm .  =  = 0 in all these scenarios.A second form of sensitivity analysis considered a range of deviations fromthe MAR specification based on subject-matter knowledge. A survey inScotland specifically sampled harmful and dependent drinking in-patients andout-patients attending alcohol addiction services in two Edinburghhospitals, finding an estimated mean weekly consumption of 198 (95% CI:185–211) units.M = 70bootstrap samples to be imputed, based on the fraction of missing information of 70%.39The SHeS had an overall survey response level of 60% and a proportion of consentto record linkage in Stage 1 of 0.91 with  = 0.001135, and using the first bootstrap sample, = 0.001021. Since also  = 0.546,  = 0.454, equation socio-demographic composition and health outcome probabilities , 2 and 3A total of 4903 participants (91.1%) were classed as current drinkers with theremaining 478 participants (8.9%) considered non-drinkers (ex-drinkers orlifetime abstainers). Mean weekly consumption from the survey-weighted estimateswas 21.8 units for men and 10.8 units for women. Imputing usual weekly alcoholconsumption in Stage 3 using each of the created bootstrap sample data setsunder a MAR assumption, resulted in an estimate of 22.4 units (3% increase)among men and 10.8 units (0% change) for women  in the most conservative sensitivity analyses(MNAR3M) to 33.3 units (53% increase) in the most extreme (MNAR5M). Among women,this range was smaller with corresponding figures of between 11.0 units (2%increase) and 11.9 units (10% increase), respectively ,MNAR3UB40 our higher estimates appear to be most appropriate.Our approach forms an important additional analytic strategy for addressingnon-participation in population-sampled studies. The key innovations of our approachare the incorporation of auxiliary topic-relevant data into unit non-responsecorrection in addition to the conventional socio-demographic data, combined with thecreation of synthetic observations for non-participants and the application ofpattern-mixture modelling to explore sensitivity to plausible departures from theMAR assumption. Resultant alcohol consumption estimates were sensitive toassumptions regarding both drinking status of non-participants and consumption leveldifferences between participants and non-participants. The refined estimates werebetween 3% and 59% higher among men and up to 16% higher among women relative to theregular survey-weighted estimates. Given that survey-based alcohol consumptionestimates scale up to approximately half those indicated by sales data,41 We circumvented the challenges associated with gaining rich datacharacterising the population, and non-participants in particular, by generatingsynthetic observations for non-participants. The second strength is theapplication of the much discussed but little implemented ‘principled sensitivity analysis’33 pattern mixture modelling to optimally42 and transparently specify MNAR models.43 In cases where no delta values are obviously more realistic than others,Rubin has emphasized the need for easily communicated models43 which areparticularly valued by policymakers;45 we found it useful toimpose assumptions in order to fix upon a plausible mechanism, consideringspecific conceivable scenarios in the context of the a prioriinformation available.The first strength of this work is the utilisation of linked survey recordsenabling the extension of comparisons of participants and the general populationfrom basic socio-demographic variables to health outcomes.46 Previous work on refinement of alcohol consumption data in the presenceof non-participation has been based on Swedish data41 which has also considered the implications for impact of the use ofaugmented data on estimates of consumption prevalence but was based onretrospective alcohol-related hospitalisation data. This offers an alternativeapproach which does not rely on the attendant passage of time required forfollow-up data. This methodology alone is unable to address bias arising fromparticipants mis-reporting their alcohol consumption. It is possible to accountfor such self-reporting bias by way of incorporation of sales data, for instance.16Limitations include the possibility of distortion arising from surveyparticipants not consenting to record linkage which could explain some of thedisparities between health outcomes in the survey samples relative to thegeneral population; however, this only affects 9% of participants andpreliminary analyses suggest minimal differences between these groups (dataavailable on request) indicating that this is unlikely to greatly distortfindings. The available alcohol-related harm outcome measures were restricted tothe relatively extreme occurrences, hospitalisation and death, with no data onthe more frequent occurrences of commonplace harms related to alcohol abuse suchas nausea, cognitive impairment and missed working days. This may explain therelatively small changes seen in the MAR estimates despite largesurvey-population differences in alcohol-related harm.47 based on health outcome statuses as well as socio-demographiccharacteristics. It is not clear how to implement MNAR methods witheasily communicated models in these approaches.As an alternative to our procedure of generating syntheticobservations and implementing MI, we could have applied weights ortaken a Bayesian-based approach48A possible alternative to creating multiple data sets of syntheticnon-participants followed by single stochastic imputation on each isa nested MI procedure where more than one final imputed data set isgenerated for each first-stage imputed data set. This could becomputationally efficient if stage 2 was very slow and stage 3 wasrelatively fast, which was not the case here, and could help topartition the fraction of missing information between stages 2 and3, but would require alternative combining rules to Rubin’s.(1) Otherwithin-survey proxy non-participants: e.g. those withother risky health behaviours such as heavy smoking: this may notform a useful reference point when considering plausibility of deltavalues; (2) Expert opinion:49 we canvassedthe broader international alcohol research community through amailing list (administered by the Kettil Bruun Society) toinformally elicit expert opinion; no useable information was gainedfrom this channel; (3) Record-linked cohort data:the use of baseline alcohol data on cohort study subjects includingthose who drop out during follow-up (taken as proxies fornon-participants on the basis that they may be somehow similarly‘disengaged’) and those remaining in the study follow-up (acting asthe corresponding counterparts for survey participants);50 no such suitable data could be identified; (4) Retaildata: such external sources of information could beused as the basis a plausible upper bound for population meanconsumption; (5) Worst-case bounds making no assumptionsabout the missing data: completely assumption-freeapproaches, which consider all feasible values of the missing data,generate exceedingly wide bounds for continuous measurements likealcohol and are thus often not directly useful for policy purposes;approaches to narrow such bounds often rely on instrumentalvariables or longitudinal survey data which were not available inthis case.51The assumptions and relative merits of our approaches to determiningdelta values for the pattern-mixture approach are inherentlyuntestable, and there is an array of alternatives to the propensity-and literature-based scenarios, including: The following considers possible alternatives approaches in specific steps of theanalyses: National survey data are crucial resources for quantifying and monitoring trendsin health related behaviours with information used for the development,implementation and evaluation of social and public health policy. As such,methodological improvements are of interest to a wide international audience ofpolicy makers and researchers. The development of an effective post-hoccorrection procedure for ever-worsening non-response in resource-intensivepopulation-sampled studies offers an enhancement at no additional cost to datacollection. This advanced methodology will potentially be applicable to existingand future surveys wherever there is the capacity to record-link surveys withadministrative data. Presently, linkage of survey data to routine health recordsrepresents a cost-effective means of generating valuable longitudinal data butis performed in very few countries. In exploiting such linkage, our workdemonstrates the extended utility of record linkage, providing further impetusfor its wider uptake internationally.52 However, possible ethical issues related to accessingoutcome data of individuals who have chosen not to participate in a survey maymean that even in such countries stage 1 of our approach might be applicable.Regardless, stages 2 and 3 of our proposed methodology would be applicable inthese settings. Our approach to the sensitivity analyses was specific to thecontext in terms of the estimate of interest and level of participation.Different applications will require distinct approaches to be formulated. Inconsidering the most suitable derived estimates ,we were guided by overall estimates obtainable from external alcohol retaildata; dependent on the specific context of the wider applications, referenceshould be made, where possible, to such relevant sources.Synthetic generation of survey non-participants is not necessary in countrieswith unique population identifiers and comprehensive linkage (such as the Nordiccountries) with the ability to follow-up all individuals regardless ofparticipation status.The presented application suggests that non-response may contribute to thegeneral under-estimation of alcohol consumption in survey estimates. There isscope for application to other survey-derived information, which can be discrete– cigarette smoking and obesity, for instance – for which only stage 3 of ourprocedure would need amending. The outcomes of choice in our application werealcohol-related harms and all-cause mortality on the basis of their strongassociation with alcohol consumption; single or multiple outcomes can beselected and good candidate outcomes for specific applications are those whichhave the strongest associations with survey items of interest. Further,non-health external data sources such a taxation or education records could beused to provide auxiliary information to correct for non-participation bias inother research areas. Moreover, this paper describes tackling a singleincomplete variable; however, the method can be extended to multiple incompletevariables.The current method requires that the informative data for creating the syntheticnon-participants are categorical, since we are determining the missing numberswithin discrete cells. It may be possible to incorporate continuous data – suchas the number of health outcomes experienced – in a further stage by inferringthe distribution among the non-participants such that a value could be assignedto each synthetic non-participant as a draw from that distribution and repeatedacross multiple replications to allow properly for uncertainty. This would bemost appropriately performed by way of MNAR imputation to incorporateinformation about number of alcohol-related harms from the population comparisondata.53 or differential consumption-harm relationships by alcohol product type.54 The application we describe focussed on a quantity estimate but there isgrowing recognition that non-representativeness can also lead to bias inestimates of associations.55 We plan to develop, apply and test our methodology for associationestimates.Sensitivity analyses could potentially be used to address any differentialconsumption-outcome associations among area deprivation categories, i.e.allowing for the possibility of interaction effects suggested by the greaterlevels of alcohol-related harm among the more deprived for equivalent levels of consumption57 Selection modellingexpresses departures from MAR as coefficients in a logistic regression model fornon-participation on alcohol consumption and other covariates: the sensitivityparameter may therefore be less intuitive than in the pattern-mixtureframework,35 and hence less easy to relate to subject-matter knowledge.49 Shared parameter models,58 in which the measurement of interest and missingness processes are jointmodelled, offer yet another option which can be explored.A major alternative to the pattern-mixture approach to MNAR sensitivity analysisis the selection model approach.We offer a means to extend the addressing of non-representativeness in survey databeyond the use of conventional inverse probability weights by developing amethodology which harnesses administrative and record-linked data. The key advantageof our approach is the relaxing of the assumption that socio-demographicallyequivalent participants and non-participants are alike in other ways: theapplication of the MAR method to administrative health record-linked data is animprovement on the conventional application of survey weights, and the MNAR methodsutilise the best available data to make plausible assumptions about how they mightdiffer.


The expansion of national newborn screening (NBS) programmes has provided significant benefits in the diagnosis and early treatment of several rare, heritable conditions, preventing adverse health outcomes for most affected infants. New technological developments have enabled the implementation of testing panel covering over 50 disorders. Consequently, the increment of false positive rate has led to a high number of healthy infants recalled for expensive and often invasive additional testing, opening a debate about the harm-benefit ratio of the expanded newborn screening. The false-positive rate represents a challenge for healthcare providers working in NBS systems. Here, we give an overview on the most commonly used strategies for decreasing the adverse effects due to inconclusive screening results. The focus is on NBS performance improvement through the implementation of analytical methods, the application of new and more informative biomarkers, and by using post-analytical interpretive tools. These strategies, used as part of the NBS process, can to enhance the positive predictive value of the test and reduce the parental anxiety and healthcare costs related to the unnecessary tests and procedures.  There are significant long-term cost benefit advantages in diagnosing inherited metabolic disorders shortly after birth. Many conditions which if untreated are characterized by episodes of metabolic decompensation can be effectively managed through diet when diagnosis is made before the development of symptoms. An early diagnosis often prevents clinical complications that may be irreversible ,2.Newborn screening (NBS) plays a crucial role in preventive medicine. Advances in technology and more sensitive diagnostic tests allow infants with manageable congenital diseases to be identified and treated within days of birth ,4.With the advent of Tandem Mass Spectrometry (MS/MS), one of the most important advances in the field of NBS, a single dried blood spot (DBS) can be used to screen a wide range of conditions . SimultaMoreover, conditions included in national NBS programs continue to increase, also thanks to the development of novel immunoassay and real-time PCR testing platforms ,7,8.Despite the high sensitivity and specificity of current methods employed in NBS, a positive screening result does not establish a definitive diagnosis and follow-up analytical tests are required. Positive newborns must be recalled for clinical evaluation and additional laboratory tests. Given that NBS programs now include many more disorders requiring more tests than in the past, the rate of false positive results has increased .The sensitivity and specificity of NBS tests are dependent on decisions regarding cut-off values. A false negative is usually more harmful than a false positive since an affected newborn may be missed. Adjusting cut off values to reduce the risk of false negatives often has the effect of increasing the false positive rate. Screening may, therefore, lead to a high number of unnecessary recalls and additional testing which can cause a non-optimal allocation of healthcare resources and parental anxiety with long-term consequences on the child–parent relationship . An evaluation of parental stress highlighted significantly higher levels of anxiety in mothers with newborns in the false-positive group than in mothers whose infants tested negative. The mothers’ anxiety did not fully resolve after further testing eliminated the possibility of illness. Infants in the false positive group were more likely to be taken to emergency rooms and to be hospitalized than those who were not recalled at birth ,12.Given the psychological and economic impact of false positives, significant efforts have been made to improve the performance of NBS assays.The most effective diagnostic markers are specific metabolites produced by a disease which are not present in healthy people.The production of succinylacetone (SUAC) is caused by a deficiency of the enzyme fumarylacetoacetate hydrolase in the tyrosine catabolism pathway. The presence of SUAC in blood or urine is pathognomonic for Hereditary Tyrosinemia type I (HT-1). In the past, SUAC could not be extracted from DBS samples prepared according to classical protocols. Thus, the amino acid tyrosine was used as the metabolic marker for HT-1, which resulted in a high false positive rate due to the high incidence of benign transient Tyrosinemia in the neonatal population. Moreover, newborns affected by HT-1 can have normal tyrosine levels at birth, which leads to a high risk of false negative results and misdiagnoses ,14,15. To resolve this problem, hydrazine can be added to the alcoholic extraction solution, allowing the formation of hydrazone from hydrazine and a carbonyl compound of SUAC, which can be extracted from the sample matrix at the same time as amino acids and acylcarnitines . Most laAn initial out of range screening result can be due to transient conditions, genetic variants, carrier status or overlap between the healthy and diseased population. Development of effective second-tier tests (2-TT) can minimize false positives and avoid unnecessary recalls . A 2-TT A 2-TT can identify the same target as primary screening but with improved specificity thanks to separation from isomers or interfering substances, or it can screen for another diagnostic marker not included in first tier-screening. Several authors have estimated the extent to which 2-TT based strategies can reduce economic costs associated with false-positives. Gavrilov and colleagues report that in the absence of a 2-TT program, the total average cost of the first instance of follow-up for an abnormal C3-carnitine or elevated methionine is approximately $1500 per false positive . The NatCompared to chromatography-MS/MS, flow injection-MS/MS analysis (FIA-MS/MS) for expanded NBS has important advantages. It is fast, cheap and allows for high-throughput screening. However, it is unable to differentiate isobaric isoforms. The application of a 2-TT, using chromatographic separation prior to mass spectrometric detection, can be a useful in confirming a positive first-tier result, before the newborn is recalled.A perfect example of how isobaric interferences may affect screening performance is in the combined detection of several branched-chain amino acids (BCAAs) in maple syrup urine disease (MSUD). Babies with MSUD accumulate leucine, isoleucine, valine and allo-isoleucine in blood and tissues with devastating effects . Early dThe routine screening method measures several isobaric species of different amino acids  combined in one analytical signal named “X-Leu”. However, this combined signal can be elevated in newborns receiving parenteral nutrition containing BCAA or in the clinically benign condition of hydroxyprolinemia and so 2-TT is necessary to quantify allo-isoleucine, a pathognomonic marker of MSUD disease. In healthy newborns allo-isoleucine is undetectable so affected babies can be clearly distinguished, meaning that unwarranted follow-up tests to rule out MSUD are unnecessary . Unfortunately, NBS can fail to detect the intermediate form of MSUD, which often only manifests in biochemical alterations during metabolic decompensation. Although the introduction of 2-TT can increase sensitivity by lowering the X-Leu cut off, some affected children cannot be identified at birth ,25. An increase in C5-acylcarnitine (C5) levels in an NBS acylcarnitine profile can suggest both isovaleric acidaemia (IVA) with elevated isovalerylcarnitine or short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) with elevated 2-methylbutyrylcarnitine (2MBC). While the latter is an asymptomatic condition not included in worldwide NBS panels, IVA is a serious inherited condition caused by a defect in the catabolic pathway of leucine.Several papers report on an increased recall rate in newborns due to elevated C5 caused by the intake of pivalic acid derivatives. Pivalic acid contained in some antibiotics or nipple creams can conjugate to free carnitine and became a C5-carnitine isobaric compound ,27,28. IA chromatographic separation in NBS samples with elevated C5 allows for the identification of different isomers underlying abnormality before alerting the family . With 2-Not all the analytes included in expanded NBS have a pathognomonic significance and some can produce a high number of false-positive results .High levels of propionylcarnitine (C3) in newborn screening may suggest an inborn error of propionate metabolism including propionic aciduria (PA) and methylmalonic aciduria (MMA) .Children affected by PA and MMA are at risk of acute metabolic decompensation and detected newborns must be immediately recalled. Samples with elevated C3 can lead to a high false positive rate because concentrations found in affected newborns overlap with healthy individuals, reducing the marker’s specificity. However, if the C3 threshold is set too high, many affected babies can be missed .The application of a 2-TT able to identify free organic acids such as methylmalonic acid (MMA) and propionic acid (3OH-PA) on DBS has greatly improved the PPV of C3 . Sample It is not unusual for normal healthy infants to show small amounts of MMA in blood in the first days of life owing to hepatic enzyme immaturity; thus, inaccuracies in determining cut-off levels for MMA on DBS could lead, once again, to more false positives than expected. The new biomarker, heptadecanoylcarnitine C17 (C16:1-OH isobar), can be used to determine when 2-TT should be performed, even when the primary C3 marker falls within normal limits. It has been demonstrated that C17 is more sensitive than other C3 ratios in improving NBS diagnostic prediction rates .NBS laboratories use methionine as the primary marker for detecting homocystinuria and methylation disorders. Hypermethioninemia can be associated with classical homocystinuria, presenting elevated homocysteine in both blood and urine or with MAT I/III deficiency without homocysteinemia. Although evaluating homocysteine levels by a 2-TT on positive screening samples can be useful for patient management, there is no reduction in false positive rates, since an elevated methionine from an NBS always leads to patient recall. Low methionine levels can indicate an underlying re-methylation disorder, including severe MTHFR deficiency, typically associated with high homocysteine levels. Since methionine levels can be affected more than other amino acids, by poor sample storage, application of 2-TT for homocysteine quantification, when methionine on the first test is low, remains the most effective means of reducing false positives . Many scNBS for CAH, using the biomarker, 17-OH-progesterone (17-OHP), usually measured by fluoroimmunoassay on DBS, has been introduced in many states around the world. The levels of this hormone, which can be altered by the degree of prematurity and sampling time, are very variable in the first days of life, with the consequence that there is a high false positive rate .Adjustments to cut-off values based on different gestational ages and/or on birth weights can help reduce the number of false positives. Moreover, as 17-OHP decreases over time in healthy newborns but increases in affected infants, some states have added a second screening test for all newborns at 8–14 days of age . FingerhSevere combined immunodeficiencies (SCIDs) are rare disorders, characterized by a congenital defect of the immune system, associated with poor prognosis if there is delay in diagnosis and treatment. In recent years, following the development of rapid and more accurate diagnostic methods together with more effective treatments, screening for SCIDs has been incorporated into many NBS programs. So far, the most sensitive and specific method for SCIDs screening is a quantitative polymerase Chain Reaction (PCR) to dose the copy number of T-cell receptor assay gene excision circles (TRECs) . This meTwo SCIDs, adenosine deaminase deficiency (ADA-SCID) and purine nucleoside phosphorylase deficiency (PNP-SCID), are characterized by enzyme defects in the purine salvage pathway, resulting in the accumulation of metabolites that are toxic to lymphoid lineage cells. Consequently, these two immunodeficiencies are also metabolic disorders and the increased production of purine metabolites can be identified by mass spectrometry on DBS. This method has the advantage of being cheap—the biomarkers can be included in the analyte panel during routine expanded newborn screening—and can thus be adopted where there are not sufficient funds for genetic screening by TREC testing.It has been demonstrated that deoxyadenosine and deoxyguanosine  are absent in healthy subjects. The test has a very high sensitivity and false positive results can be avoided by a prompt second-tier LC-MSMS method, able to separate any interferers ,44.In the last fifteen years, screening for several LSDs has been included in several NBS programs. Analysis is performed by measuring lysosomal enzymatic activities on DBS using either MS/MS or fluorimetric assay . RecentlSome ethical issues about expanding NBS for LSDs remain unresolved. One of the main problems is a lack of genotype-phenotype correlation. Many genetic alterations detected in lysosomal NBS programs have been associated with late-onset forms or have been identified as genetic variants of unknown significance (VUS). Moreover, the high frequency of pseudodeficiency alleles increases the rate of false positive results. To reduce unnecessary recalls some NBS laboratories carry out genotyping using residual DBS specimens for 2-TT analysis . HoweverNevertheless, the efficacy of using biochemical biomarkers for LSD has yet to be clarified; data is lacking on biomarker concentrations in newborns with attenuated phenotypes and further studies must be conducted.Worldwide NBS programs for Pompe disease (PD) are based on performing assays for enzymatic activity of acid α-glucosidase (GAA), one of the GAA isoforms.The main cause of false positive results in Pompe NBS is the high prevalence of pseudodeficiency alleles in the newborn population. Pseudodeficiency variants decrease enzyme affinity on the artificial substrate used in testing, but have no effect on the natural substrate. In Taiwan, where the Pompe NBS program employs fluorescence assay, screening results have shown a high frequency of the pseudodeficiency allele p.G576S (14.5%), making the false positive rate unacceptable. To improve NBS performance, the ratio of neutral and acid α-glucosidase activities has been proposed as a cutoff to distinguish between true-positives and false-positives. Unfortunately, however, cut off values cannot distinguish between newborns with the milder form of PD and pseudodeficit carriers .Recently, Tortorelli and colleagues have described a new biomarker employed in NBS for PD based on the measurement of the creatine/creatinine (Cre/Crn)/GAA ratio on DBS . QuantifGenetic testing on positive NBS samples for Fabry disease (FD) has revealed a high prevalence of neutral variants in the GLA gene. Whereas the diagnosis of male patients with the classical phenotype is usually straightforward, the clinical implications of GLA variants of uncertain significance remain incompletely understood .In 2008, the deacetylated form of globotriaosylsphingosine (LysoGb3) was reported as a prognostic biomarker for FD . All malIn the light of these findings sensitive analytical methods by LC-MS/MS have been developed to measure Lyso-Gb3 on DBS from FD patients . Males wThe deficient activity of α-iduronidase (IDUA), one of the lysosomal enzymes involved in the breakdown of glycosaminoglycans (GAGs), leads to the accumulation of heparan sulphate (HS) and dermatan sulphate (DS) in affected MPSI patients also before birth . The earIn many NBS laboratories, second-tier genotyping analysis is carried out for newborns with low IDUA activity. However, because of the high frequency of private mutations and VUS, molecular analysis may fail to predict the clinical phenotype of pre-symptomatic newborns.The application of LC-MS/MS technology to quantify DS and HS has allowed the development of a 2-TT on DBS samples to confirm or overrule first tier results and improve the diagnostic process ,63,64. GThe NBS program for Gaucher disease (GD) is based on the measurement of lysosomal β- glucocerebrosidase (GBA) activity using tandem mass spectrometry or fluorimetric assay. In affected patients, GBA deficiency causes the accumulation of glucosylceramide in lysosomes of macrophages, called Gaucher cells.Chitotriosidase and CCL18 are well-known Gaucher cell markers but they are non-specific and can also be elevated in other lysosomal storage disorders such as Niemann-Pick C1. Researchers have focused on discovering new biomarkers for the disease. High levels of glucosylsphingosine (lysoGb1) have been found in plasma samples of symptomatic GD patients . FurtherIn 2006 newborn screening for Krabbe disease (KD) began in New York State to identify pre-symptomatic cases of the infantile disease and ensure prompt treatment. As previously described, the first-tier test is based on measuring the enzymatic activity of galactocerebrosidase (GALC) which plays an essential role in myelin turnover .Genetic testing conducted on newborns with low GALC levels may not be informative due to the large number of non-disease-causing polymorphisms as well as high rates of VUS and pseudodeficiency variants .To reduce the rate of false positives, a LC-MS/MS 2-TT has been developed to analyse psychosine concentrations on positive DBS samples. Several authors have reported higher levels of this biomarker in the DBS samples of infantile KD patients identified through NBS than in samples of newborns with normal GALC activity ,75,76.The combination of 2-TTs and post-analytical tools can reduce the incidence of false positives. Ratios of GALC activity to the other lysosomal enzyme activities are informative because they can distinguish true positives from true negatives .Several marker ratios are useful in the interpretation of NBS results. In screening for Phenylketonuria, the phenylalanine/tyrosine ratio has a greater clinical sensitivity and specificity than phenylalanine concentration alone . This isThe C3/C2 ratio is another powerful indicator able to enhance the predictive performance of the MMA screening test. This ratio is usually used as a secondary parameter with a greater prognostic value than C3-carnitine levels alone. However, cutoff setting remain problematic. Low cutoff levels can result in large numbers of false positives while high cutoffs levels may lead to affected newborns being missed. Indeed, several affected newborns have been missed because both C3 and the C3/C2 ratio were within established normal ranges. The best approach is to lower cutoff levels so as not to miss affected newborns, while using 2-TT to reduce the number of false positives.C8/C10 and C8/C2 ratios together with C8-carnitine levels are commonly used for predicting an MCAD diagnosis based on NBS results. The use of these ratios can aid the interpretation of milder alterations in acylcarnitines with medium chain. The acylcarnitine profile of severe MCAD deficiency is unequivocally recognizable but mild forms are less easy to identify.Advances in the field of NBS have spurred the development of data collection platforms for the interpretation of metabolic profiles. Many screening laboratories used Region 4 Stork (R4S), active between 2004–2013 to interpret screening results.R4S was developed to improve how screening results are interpreted by integrating abnormal values with additional clinical data. Analyte ranges for each pathological condition were established which were more informative than the single cutoff values used by individual laboratories . The posThe advanced version of R4S, Collaborative Laboratory Integrated Reports (CLIR) extends the previous multivariate pattern-recognition software, by incorporating additional demographic information such as age, birth weight and gender which can be responsible for significant discrepancies among results collected by different labs .CLIR collects large numbers of normal and positive screening profiles from NBS programs worldwide. The platform provides post analytical tools not only for the expanded NBS of metabolic disorders but also for other NBS programs. The availability, on a single platform, of data for all types of disease-related analytes including secondary biomarkers and analyte ratios as well as data on clinical variables has led to significant improvements in false-positive rates .The extraction of DNA from DBS allows for molecular testing in NBS programs . MoleculGALT mutations (seven mutations and two variants), to improve the specificity of NBS for Galactosemia. The approach seems useful for newborns with initial positive screening results, but it needs to be proven in prospective studies [Second-tier molecular testing was first introduced for conditions such as cystic fibrosis and sickle cell disease ,83. Scre studies . In the  studies .However, although this strategy can be used for a wide range of conditions, there remains some doubt over its application in disorders associated with acute metabolic decompensation. The potential benefit of a second-tier genetic test must be balanced against the potential harm in delaying recall for time-critical conditions.Next-generation sequencing (NGS) technology allows sequencing of the whole genome, the whole exome or of a targeted gene panel to be performed. For inherited disorders, the first line of testing is usually a targeted gene panel that focuses on a specific cohort of genes. The feasibility of NGS as a second-tier diagnostic test in NBS has been studied .Second-tier NGS testing using DNA from the same DBS after primary screening of TRECs was proposed also for severe combined immunodeficiency and a targeted next generation approach was considered as an available second-tier approach for Pompe disease when enzymatic activity on DBS is below the established cutoff values ,86. A feHowever, next-generation DNA sequencing technology raises several ethical considerations because of the detection of variants of uncertain clinical significance, the identification of carrier status and pseudodeficit or benign conditions without clinical manifestations . InterprOther limits of these technologies are the high costs, the prolonged turnaround time and the identification of misclassified variants.NBS has changed the natural history of several diseases and improves the quality of life for many patients. Effective NBS programs can bring significant cost-benefit advantages for public healthcare systems, but a high incidence of false positive results can cause serious and lasting psychological consequences for newborns and their families. It is, therefore, essential to develop more specific assays to investigate altered biomarker levels in primary screening results and to eliminate as many false positives as possible. The application of two or more strategies to improve the consistency of outcomes in NBS programs involves costs that can be well sustained by public health systems. Unfortunately, few NBS tests give a PPV near 100% but technological advances in methods are leading to important improvements. The aim of healthcare providers working in NBS systems should be to improve the positive predictive values of screening tests by applying appropriate analytical and post-analytical tools.NBS programs should contain costs, prevent parental anxiety arising from results which are inconclusive or of unknown clinical significance and reduce the time needed to make a diagnosis.