Blood transfusions are one of the most common procedures performed in hospitalized patients. Yet, despite all of the measures taken to ensure the safety of the blood supply, there are known risks associated with transfusions, including infectious and noninfectious complications. Meanwhile, issues with blood product availability, the need for compatibility testing, and the storage and transport requirements of blood products, have presented challenges for the administration of blood transfusions. Additionally, there are individuals who do not accept blood transfusions . Therefore, there is a need to develop alternative agents that can reliably and safely replace blood. However, although there have been many attempts to develop blood substitutes over the years, there are currently no such products available that have been approved by the United States Food and Drug Administration (FDA). However, a more-recently developed hemoglobin-based oxygen carrier has shown promise in early clinical trials and has achieved the status of “Orphan Drug” under the FDA. SUMMARYIntroductionBlood vs. blood substitute: not one and the samePerfluorocarbon-based blood substitutesHemoglobin-based blood substitutesPEGylated hemoglobinsConclusion 1-3. Nevertheless, the risks of blood transfusion cannot be entirely eliminated. Moreover, some recognized risks have proven to be quite challenging to prevent or mitigate, such as iron overload (a result of receiving repeated transfusions of red blood cells [RBCs] which contain 200-250 mg of iron) and transfusion-related acute lung injury (TRALI), a complication in which acute lung swelling (pulmonary edema) classically occurs as a result of antibodies to donor human leukocyte antigen (HLA) in a susceptible patient though alternative pathways have also been described 4,5. Meanwhile, there are well-known transmissible disease risks that are not or cannot be tested for; these include malaria transmission, transmission of human granulocytic anaplasmosis (formerly erlichiosis) bacteria, and transmission of prion disease , to name just a few of these risks6-8. Emerging, yet to be recognized, risks are also a major concern of blood transfusion.Although the search to discover a safe and effective blood substitute, commonly referred to as “artificial blood” but more scientifically termed “oxygen therapeutic agent” (for reasons described later in this article), has been ongoing for a number of decades, true success has yet to be realized. Blood transfusion is still one of the most commonly performed procedures in hospitalized patients nowadays, and the safety of blood products has increased for several reasons: 1. enhanced health and risk-factor screening of prospective blood donors, 2. increased donor testing for transmissible diseases such as human immunodeficiency virus (HIV) and viral hepatitis (hepatitis B and C viruses) but now also including testing for more-recently recognized transmissible disease risks , and 3. improvements in the processing of blood products h, present in less than 1% of the world’s population).Availability of blood products and compatibility present additional challenges in the practice of transfusion. Seasonal blood shortages, particularly during the height of the summer and winter holidays, are not uncommonly encountered throughout regions in the United States, sometimes causing elective surgeries to be postponed. Furthermore, there can be great difficulty in finding available blood for patients who are highly immunized  or for those who have a rare blood type  or other reasons . For this and all of the above reasons, development of a universal blood substitute would seem like the ideal solution. Nevertheless, the development of such a product has proven to be quite elusive despite many attempts to overcome the barriers to success. This article will further discuss these attempts, the products that were developed and tested, and the failures that occurred. Finally, it will highlight promising newer blood substitutes that are currently undergoing testing, known as PEGylated hemoglobins. It should be emphasized that although the authors attempted to comprehensively cover all known developed products, it is highly likely that some products are not discussed in this article.9.Blood, often equated to the substance of life, is involved in a number of important physiologic functions owing to its multiple plasma components and cells. Naturally, blood is central to providing tissues with oxygen and nutrients as well as waste removal. However, circulating white blood cells (granulocytes and lymphocytes [B- and T-cells]) carry out important immune functions while platelets, plasma coagulation and fibrinolysis factors are necessary for the balance of blood clot formation and degradation. Hormone transport is yet another important function of blood. Perhaps less well known is that blood also plays a major role in pH buffering, since the blood pH must be tightly regulated around pH 7.401.Blood substitutes, on the other hand, are not really a complete substitute for blood as their name would imply, considering that such agents are merely designed to support just one therapeutic function of blood, namely, oxygen transport to the tissues. For this reason, blood substitutes are more appropriately termed “oxygen therapeutic agents” (OTAs)10. Their study was able to demonstrate that mice can survive when immersed in an oxygenated PFC solution. PFCs are a synthetic molecule composed of carbon and fluorine atoms. The interaction of these atoms forms a long strong bond that protects it from chemical degradation with fluorine ions forming an electronegative shield around the molecule as seen in Figure 1. Due to the hydrophobic qualities, a complex procedure was created to stabilize them in emulsions for intravenous use. When made into this emulsion, PFCs are able to dissolve gases better than most liquids. This is due to fluorine’s low polarizability which decreases the van der Waals interactions between the PFC molecules. These interactions are known to keep together nonpolar molecules. PFCs, in turn, have strong intramolecular bonds making them stable, but they have very weak intermolecular bonds allowing them to behave as gas-like fluids which easily dissolve other substances of low cohesivity . The rate of uptake and release of O2 by PFCs is unaffected by temperature and environment. In contrast to hemoglobin, which relies on localized chemical bonds for the dissolution of O2, PFCs follow Henry’s law that dissolved O2 concentration at equilibrium at a given temperature is directly proportional to the gas’ partial pressure, resulting in the rapid and extensive extraction of O2 when needed. The practicality of PFCs being a good candidate for in vivo use comes from its inert qualities to dissolve gases, its combination of O2/CO2 solubilities, and its molecular stability.The ability for perfluorocarbons (PFCs) to be used as oxygen carrying agents was first described by Clark and Gollan in 196611.The exceptionally strong intramolecular binding and low intermolecular cohesiveness of liquid PFCs result in unique properties that make them different from other regular organic compounds. Fluorinated compounds can reach a level of effectiveness in performance that cannot be achieved by non-fluorinated compounds. In summary, fluorinated compounds such as PFCs offer unique combinations of properties that can make them irreplaceable and constitute the basis for potential biomedical applicationsTable 1). The chemical composition of Fluosol was a 7 to 3 ratio of perfluorodecalin and perfluorotripropylamine while using pluronic F-68  as an emulsifier. It was available as a 20% solution with the ability to carry 7.2% volume of 100% O2 at 37°C. This compound also could carry 34% of the O2 content of whole blood at a hemoglobin level of 14 g/dl12. The first use of this commercially-available product was reported in Japan with infusion of 500 ml and 1000 ml given to patients with severe gastrointestinal bleeding and surgery-related blood loss for esophageal cancer, respectively13,14. Subsequent in vivo and in vitro clinical trials after the initial use in Japan examined Fluosol use in a multitude of patients with different clinical issues such as severe anemia, peripheral vascular disease and extensive blood loss from a surgical wound while also being tested in patient population that normally refused blood products . These published trials were able to demonstrate that Fluosol, the first widely available PFC emulsion, had its merits with a so called “proof of principle” as an injectable tissue oxygenation agent. Over 20 years, the use of Fluosol in laboratories throughout the world allowed for an extensive review of PFCs and was important in the development of concentrated, room temperature-stable, second-generation PFCs.The first generation of PFC emulsions to be developed was Fluosol-DA , which was engineered over 30 years ago  is a stable second-generation concentrated PFC. The original formulation consisted of 60 grams PFC/dL with two active ingredients: perfluorooctyl bromide and perflubrodec. The latter was added in small quantity to stabilize particle growth during storage15. To make this product, an egg-yolk phospholipid was used as an emulsification agent for PFCs in a buffered electrolyte solution. The final product had an approximate 24-month shelf life when stored between 2 - 8oC but was stable enough to tolerate exposure at room temperature for a few weeks. Over 250 preclinical studies were performed to understand the safety of Oxygent in various animal species before moving on to studies that demonstrated efficacy in humans. Of note, toxicology studies have shown that the emulsion is well tolerated without severe adverse effects when given at appropriate clinical dosing (1.0 - 6.0 ml/kg). In Europe, a large phase III multicenter study showed that Oxygent in conjunction with hemodilution decreased the need for RBC transfusion in 492 patients undergoing non-cardiac surgery16. Patients were randomly distributed into two groups: included in the control group were patients that were transfused intraoperatively at a hemoglobin concentration of less than 8.0 g/d while in the test group, patients first underwent acute normovolemic hemodilution to a hemoglobin of 8.0 g/dL then were dosed with Oxygent. Results showed that 26% of patients in the test group had a reduction in the number of RBC units transfused when compared with the control group (16%). However, in the United States. a second trial was terminated because of possible increase in stroke rates in the test arm17.Oxygent18. Unfortunately, at this time there are no published data available from this trial, but the company did claim that the initial goal of increasing the partial pressure of arterial O2 levels was met. Again, like Oxygent, a second trial was suspended19.Oxycyte™  is similar to Oxygent as an emulsified compound with purified egg yolk phospholipids composed of C10F20 PFCs. This product was initially tested in phase II trials where patients who had traumatic brain injury were given the emulsified product2 per 100 ml. This in turn required patients to receive supplemental O2 because most of the O2 carried by the substance was unloaded before reaching the microvasculature. Another issue with the product was that it was excreted slowly with metabolites remaining in the body for months after administration. It also had biological side effects such as a temporary decrease in the platelet count and fevers. Lastly, due to the short stability time of approximately 8 hours, it created difficulties for use in coronary balloon angioplasty, the indication for which the product had gained its approval. This led the FDA to remove Fluosol from the market in 1994.PFCs are ambitious products that in theory and chemically seem to be a viable option for clinical use. However, issues with manufacturing and development have led them to become mostly discontinued. Fluosol, as mentioned above, demonstrated this “proof of principle” which led to a newer formulation being FDA approved in 1989, but it eventually suffered from clinical shortcomings. When put into clinical use, the formulation only delivered 0.4 mL of O2 delivery; patients had to receive supplemental O2 inhalation because the delivery was less than 30% of normal blood18. Oxygent use in a large multicenter study also showed more serious adverse effects/events in the PFC group vs. the control group (32% vs 21%) with a possible increase in stroke rates seen in the United States phase III trial. Most studies on Oxygent have been terminated because of costs and a suggestion that the manufacturers did not have faith in the product’s clinical or commercial success.Second-generation iterations of PFCs also had their pitfalls with OⓇ, Russian Academy of Sciences, Puschino, Russia), but, unfortunately, until a well-designed trial is performed in the United States, PFCs will not be widely used. Currently, Perftoran , is awaiting clinical trials20. Ultimately, due to the lack of clinical evidence and the timeline for approval from the FDA, PFCs are unlikely to be a viable blood substitute any time in the near future.Due to initial clinical trials with these products showing lack of beneficial outcomes, no current trials are ongoing with PFCs. There are certain products that are being used in Mexico and Russia  agents have been approved and used clinically in two countries (Russia and South Africa) outside of the United States21-23. Yet these endeavors have brought new insights into the basic biophysical characteristics of hemoglobin and oxygen transport systems and have paved the way for the development of products which may be readily available with efficient O2-carrying capacity and minimal adverse interactions with biophysical hemodynamics24,25. In that manner, hemoglobin-based products have been developed as supplements or pharmaceutical-bridging agents to allogeneic RBC transfusion.Efforts to develop alternatives to blood date back to the 17Figure 2) is a globular protein having a quaternary structure and consisting of two α and two β chains with four heme groups that are themselves made up of porphyrin rings with a central iron atom that can bond to O2. This oxygen-heme bond results in a conformational (shape) change in the hemoglobin molecule which in turn progressively increases the affinity of hemoglobin for additional O2 molecules . Thus, a small change in O2 partial pressure results in a large change in the amount of O2 bound or released by hemoglobin. A number of additional factors, such as temperature and pH, can alter the oxygen-hemoglobin dissociation curve. Similarly, 2,3-diphosphoglycerate , a highly-anionic product of the RBC glycolytic pathway and present in normal human erythrocytes (RBCs), also directly affects the binding of O2 to hemoglobin. As the concentration of 2,3-DPG rises, the oxygen-hemoglobin dissociation curve “shifts to the right”, thereby allowing the release of O2 to tissues at higher than normal O2 partial pressures.In simplified terms, hemoglobin  which leads to vasoconstriction  and the development of O2-, heme-, and globin-based radicals26. In addition, cell-free hemoglobin in the absence of 2,3-DPG is an extremely inefficient O2 transporter, capable of off-loading only a very small amount of O2 to the tissues. Owing to this highly-reactive and toxic nature of cell-free hemoglobin as well as the necessary-presence of 2,3-DPG for O2 transport efficiency, the development of HBOCs  has been quite challenging. Yet, successful development of an HBOC blood substitute is quite advantageous in that compatibility testing  is not required prior to infusion, it can be sterilized by ultrafiltration and low heat (pasteurization) to inactivate infectious agents, and could have a long shelf life, none of which are characteristic of donor RBC products.Cell-free hemoglobin can potentially be used as a blood substitute because hemoglobin maintains its ability to transport OoC27. SFH’s could be prepared by either ultrafiltration or crystallization. SFH prepared by ultrafiltration was characterized by a substantially lower content of residual membrane phospholipid and a more restricted protein composition. This preparation was also essentially free of vasoconstrictor and contractility-depressant actions on the ex vivo perfused heart. In contrast, crystallization-produced SFH resulted in a product that was less well purified of both phospholipid and protein constituents. Thus, it was likely to generate denatured protein aggregates during storage and exhibited vasoconstrictor and contractility-depressant activity which could vary significantly from batch to batch. These findings indicated that preparative methodology based on ultrafiltration and size-exclusion yielded SFHs which were superior in respect to those produced by the crystallization method21.The first generation of blood substitutes to be produced were the stroma-free hemoglobin (SFH) products. SFHs were prepared by lysis of packed RBCs forming soluble hemoglobin. This mixture was then centrifuged to remove the bulk of red cell stroma, leading to the production of SFH. The procedure resulted in 500 cc’s of a 7 g/100 ml solution of hemoglobin with normal physiologic concentration of sodium, potassium, and bicarbonate. The pH of the solution was 7.1 to 7.2, and the osmolality was 270 to 280 mOsm/kg. The methemoglobin concentration ranged from 7 to 12% of the total hemoglobin concentration which did not change significantly over a four-week storage period at 4Table 2) were pyridoxilated hemoglobin-polyoxyethylene conju-gates (PHPCs) that have been prepared through chemical modification of SFH. These products have been designed to prevent the major disadvantages of SFHs reported in multiple studies, namely, increased O2 affinity, short circulatory half-life, and nephrotoxicity17. PHPC is prepared after acquiring SFH; the free hemoglobin is pyridoxilated  to adjust the O2 affinity and later conjugated with α-carboxymethyl-ω-carboxymethoxy-polyethylene to increase the molecular weight and ensure a longer circulatory half-life. The O2 affinity of such crosslinked hemoglobin was found to be higher  compared to native human RBCs, and it remains in a deoxy (T) state with the utilization of 2,3-DPG analogs28. The second-generation HBOCs that have been developed are PolyHemeⓇ, HemopureⓇ, and HemoLinkTM. However, many of these products have been discontinued owing to the observed adverse events in clinical trials27. Hemopure, however, is approved by the South African drug council for the treatment of anemia dating back to 2001, and the product is available in the United States for qualified patients (such as Jehovah’s Witnesses who do not accept blood) with life-threatening anemia under the FDA’s expanded (compassionate use) access program29,30. In addition, one randomized, multicenter trial investigating the safety and efficacy of HBOC-201 in non-cardiac surgery patients did find that use of the HBOC product reduced RBC transfusions in 43% of patients without notable differences in mortality and serious adverse events though there was a notable excess of non-serious  adverse events associated with HBOC-20131.The second-generation HBOCs . These developed products were more homogenous to support the major toxicological and physiological experiments and also have O2 affinity similar to that of blood32. The products are the result of very-well documented steps presented by the LAIR and Baxter International Corporation29,30,33,34. They were made using outdated human or bovine blood, washing the RBCs with sterile saline to remove all the traces of plasma, and then subjecting them to hypertonic lysis. The remaining membrane material was filtered out, allowing for the purified hemoglobin to be put towards the crosslinking reaction and resulting in the purified crosslinked hemoglobin with an overall yield of 55% to 58%29,35. To maintain specific crosslinking between lysine 99α residues, an allosteric effector  was added to keep the hemoglobin in the deoxygenated (T) state. In the final stage, the reagent DBBF was added; this mixture was then heated to remove unreacted hemoglobin as well as pathogens. The final product was formulated in either Ringer’s lactate or Ringer’s acetate and could be stored frozen at -20oC for up to one year32. These products, however, lack the ability to regulate the oxidative state caused by iron in their heme group, and their phase III clinical trials have shown increased mortality with their use29. Nevertheless, Hemolink™ a noteworthy and relatively newer intermolecular crosslinked hemoglobin with activated sugar, O-raffinose combination, has been more recently developed by Hemosol Inc. The biophysical properties include oxygen-carrying characteristics (P50 of 30–40 mmHg) with non-cooperative behavior (Hill coefficient (n) = 1, compared to 2.5–2.9 for normal human hemoglobin). The product has been tested in phase I and phase II clinical trials in patients undergoing elective coronary artery bypass graft surgery, (CABG), yet no phase III trial data is available publicly36-38.The third-generation blood substitutes include hemoglobin crosslinked between the α chains with bis fumarate (DBBF) or αα-hemoglobin. This product was developed at the Letterman Army Institute of Research (LAIR) in San Francisco, CA. Similarly, Baxter International Corporation  developed HemAssist36. NO regulates endothelial mechanisms of smooth muscle relaxation by limiting the production of a vasoconstrictor known as endothelin. HBOCs are carried in the plasma and therefore free to cross through the endothelium, thereby this free hemoglobin has the potential to consume larger amounts of NO to regulate the vascular smooth muscle tone37. Other proposed mechanisms of HBOC-induced vasoconstriction include the release of norepinephrine from adrenergic regulation of the peripheral nerves and neurally-mediated distortion of chronotropic and inotropic myocardial responses as well as the disturbed neural response in maintaining smooth muscle tone39. Vasoconstriction to a certain extent has been noted to be less severe in polymerized hemoglobin preparations39-41. The phenomenon of vasoactivity and dysregulated smooth muscle tone may manifest clinically in the form of gastrointestinal distress, flu-like symptoms, and nephrotoxicity42,43.As mentioned above, several serious adverse events have resulted in premature termination of HBOC clinical trials. The following is a succinct description of these key limitations. Vasoconstriction leading to tissue hypo-oxygenation and subsequent systemic hypertension and pulmonary hypertension is the most feared limitation of HBOCs. Studies have shown that NO-scavenging by free hemoglobin has been the major factor mediating vasoconstriction44. The iron in free hemoglobin can oxidize to form methemoglobin (HFe3+) which reacts with NO to disturb the regulation of smooth muscle tone. Also, HBOCs can induce dysregulation of other physiologic functions at the vascular endothelium by extracellular hemoglobin. Free hemoglobin gets oxidized and produces unneutralized H2O2 which oxidizes ferrous and ferric hemoglobins to Fe (IV)-ferryl hemoglobin and oxyferryl hemoglobin, respectively. Ferryl hemoglobin can react with H2O2, yielding free iron and other heme degradation products. Fe (III) hemoglobin produced during hemoglobin autoxidation also readily releases heme, an additional source for oxidative stress and oxidative reactions in the plasma. The proinflammatory effects of heme and oxyferryl have been shown to also increase the risk of further oxidative stress45. Methemoglobin is also the degradation product of hemoglobin, production of which is physiologically held in control by the enzyme methemoglobin reductase in the erythrocyte. Therefore, HBOCs have the propensity of producing highly-oxidative free radicals, capable of inducing cell damage and halting other biochemically essential processes46.Substances do not normally cross the intact blood-brain barrier unless there is injury or a diseased state in which the sanctity of the blood-brain barrier is compromised. For example, in the case of head injury, cerebral hemorrhage, or ischemic injury to the brain, free hemoglobin in the HBOCs then may cross the barrier and act as a potential neuro-excitatory toxin. In addition, HBOCs may cause reperfusion injury as a result of their high O2 content47.Compared to RBCs, HBOCs lack the remarkable free-radical scavenging system which includes enzymes superoxide dismutase and hemoglobin reductase. Moreover, free hemoglobin can participate in a number of reactions that could potentially produce toxic free radicals48,49. In addition, their essential features are increased O2 affinity (reduced P50), minimal NO scavenging, and increased molecular size and chemical homogeneity50.The biochemical and physiological drawbacks of previously studied generations of OTAs have paved the way for the development of a substance that can bypass the toxic chemical and biophysical interactions of the synthetically-derived blood products, including the vascular tone and oxidative cellular damage. The proposed mechanisms of such toxic interaction have been described above and elsewhere in detail. Based on the preclinical and clinical studies, researchers are able to formulate hemoglobin molecules which are physiologically designed and tuned in such a way to exert minimal vasoactive effects to the microcirculationFigure 4) hemoglobin derived from either human or bovine sources is further modified with maleimide  or carboxylated  to produce a hemoglobin with unique O2 transport functions (Table 3). After multiple failed clinical trials of earlier generation HBOCs, it was clearly evident that these products are not clinically or biochemically identical to human hemoglobin, and due to this major drawback, it was difficult to get regulatory approval for additional large-scale human studies. With these insights, the research community shifted its focus towards developing an O2-bridging agent which can be utilized in situations where either blood is not available or where there are other limitations or contraindications for RBC transfusion. In particular, these bridging agents may be advantageous to prevent or treat ischemia-related morbidity and to potentially reduce mortality and thereby demonstrate compelling efficacy to satisfy the regulatory requirements for further studies in acute clinical settings. As a result, usage of the term HBOC was discouraged in the medical literature and the new term, “oxygen therapeutic agent” (OTA), was coined51.PEGylated  and has reduced cooperativity (Hill coefficient) and Bohr Effect (pH- dependent O2 binding) properties, the reason for which is not well understood. These physical properties render MP4 more homogenous while the molecular design prevents its dissociation into tetramer-dimer fractions which ultimately renders it free of inducing renal tubular damage. In addition, with its increased oncotic pressure, the vascular volume remains preserved. As a general rule, the larger molecules tend to be less vasoactive, and the larger size of MP4 keeps the hemoglobin molecule in the plasma where it has decreased propensity of NO scavenging comparatively to the hemoglobin in the interstitium, which is another means of limiting vasoactivity and hypertension. Based on the study results of Vandegriff and colleagues, the autoregulatory vasoconstriction induced by cell-free hemoglobin is secondary to its high P50 and increased diffusion50. The unique molecular design of MP4 allows for hemoglobin O2 to be maintained in its low P50 state that facilitates uniform diffusion of hemoglobin along its saturation gradient in the vascular channels with ultimate offloading of O2 taking place in the capillaries, which is dependent on the concentration gradient between the capillaries and the perfused tissue. Vandegriff et al. predicted MP4 to be most beneficial in situations where end organ tissue PO2 is low and with limited perfusion such as in the states of shock or traumatic ischemia50. The clinical efficacy and safety profile of MP4 was assessed in a multicenter study which supported the promising biochemistry of the molecule. The study did not report any clinically-significant adverse events with less frequent hypotensive episodes, though a transient rise in liver enzymes was noted, in patients undergoing major orthopedic surgery42. The study posited that MP4 may be of most use in clinical settings where swift O2 delivery to the ischemic tissues is necessary48,53. Several phase II studies in the United States and phase III studies in Europe were undertaken; however, the study data regarding the efficacy and clinical safety of the product has not been reported and to date, no official statement from the manufacturer has been publicized54.Hemospan, also referred to as MP4, is a human hemoglobin-based agent that was developed to mediate the vasoactivity and hypertensive effects of previously developed PEGylated hemoglobin molecules. Acharya and colleagues have thereby developed an intermolecularly crosslinked hemoglobin with surface conjugation of maleimide-activated 5 kDa PEG to surface thiol groups45. PEG-COHb has several modes of actions rendering its multifaceted therapeutic utility beyond anemia into indications that include early brain injury and delayed kidney graft function, where inflammation plays a pivotal pathological role, as well as in indications such as sickle cell disease where the inflammation and hypoxia contribute to the disease progression and associated comorbidities such as vaso-occlusive crisis55-58. The design characteristic of PEG-COHb is a modified bovine-source hemoglobin which is polymerized and carboxylated with a potential of rapidly releasing CO and increasing levels of whole blood carboxylated hemoglobin stores to 5.3 to 4.9% in in vitro rat models. This enables O2 binding to the CO sites and ultimate end organ tissue oxygenation under hypoxic conditions with the average P50 of 5 mmHG59.Sanguinate is a PEGylated and carbon monoxide-hybridized (PEG-COHb) OTA with modes of action that may be helpful to mitigate the limitations of the earlier generations of HBOCs. This novel OTA is currently being extensively studied for its characteristics vasodilatory and non-inflammatory properties including the potential to induce minimal to negligible reperfusion cellular injury54. The in vitro results suggest PEG-COHb has the potential to improve sickle cell morphology by reverse sickling, and current phase II trials are currently underway examining the value of PEG-COHb in sickle cell vaso-occlusive crisis, acute chest syndrome, and hyperhemolysis where indexed patients are treated with PEG-COHb under compassionate use. Also, there are reports of PEG-COHb increasing non-specific troponin I levels in sickle cell patients; however, there was no laboratory evidence of cardiac ischemia in over 200 patients who received PEG- COHb on a compassionate basis60. Animal studies, however, support the clinical role of PEG-COHb in instances of myocardial ischemia in rats and support its effectiveness in reducing infarct size if administered after the occlusion of the left anterior descending artery or at the time of reperfusion46. Sanguinate has been granted orphan drug status for treatment of sickle cell disease by the FDA61,62.Carboxylation of endogenous hemoglobin through the enzyme hem-oxygenase had proven to be of clinical value where oxidative and immune mediated tissue damage is feared. Similarly, synthetically derived CO-releasing molecules have the potential to be more cytoprotective and pleiotropic with remarkable oxidative stress-reducing properties. Studies have demonstrated the anti-inflammatory properties of PEG-COHb with a decrease in inflammatory cytokine levels in patients with acute subarachnoid hemorrhage and in patients with acute sickle cell crisis2 and NO. The design characteristics and physical properties of ErythroMer have preserved the hemodynamic and cellular interactions with the physical properties exhibiting its extended dry storage benefits signifying the implications for its portability and use63. Although previous clinical trials ended with a setback, the lessons learned from those studies have impacted greatly towards the optimization of the developed products64. Among a few products that have shown promise in the initial phase of development are Hemo2LifeⓇ , a lugworm hemoglobin extract which is not packed into erythrocytes or other membranes. Hemo2life has natural superoxide-dismutase-like activity that may mitigate the oxidant burden attributable to HBOCs. The product has been studied for prolonging storage of transplant organs and improving post-transplant graft function65. Other investigational HBOCs currently in the initial phases of development are OxyVitaⓇ , a stroma-free cross-linked bovine hemoglobin, HbVesicles , a liposomal-encapsulated human hemoglobin, and HemoAct , a human hemoglobin that is linked with human albumin molecules66-68.The wisdom from the lessons learned from the failed trials of OTAs has rekindled the interest of researchers and regulators enough to undertake further development of new agents. Among the most promising new OTA is the product ErythroMer , which is in the initial stages of chemical development and animal testing. The product has a strong research focus emulating normal RBC physiologic interactions with O2 delivery to the tissues. Yet, the future does hold promise as new agents, particularly hemoglobin-based agents, are already in the pipeline and one agent has achieved FDA orphan drug status for the treatment of sickle cell disease.Although there has been much interest in developing a blood substitute spanning many decades, development of a successful OTA has not been fruitful to date. This lack of success is perhaps as much attributable to toxicities related to the agents in development as it is to the physiologic nuances of O◊ Blood substitutes , better termed as oxygen therapeutic agents (OTAs), have been in development for many decades◊ The development of OTAs has taken two main approaches: 1. perfluorocarbon-based substitutes and 2. hemoglobin-based oxygen carriers◊ Currently, there are no Food and Drug Administration (FDA)-approved OTAs given the toxicities of these agents, though some OTAs are used clinically outside of the United States◊ It is possible to use OTAs in the United States via the FDA expanded (compassionate use) access program for selected patients with severe life-threatening anemia◊ There are promising new developments in the search for a safe and effective OTA


Because compensatory changes in brain activity underlie functional recovery after brain damage, monitoring of these changes will help to improve rehabilitation effectiveness. Functional near-infrared spectroscopy (fNIRS) has the potential to measure brain activity in freely moving subjects. We recently established a macaque model of internal capsule infarcts and an fNIRS system for use in the monkey brain. Here, we used these systems to study motor recovery in two macaques, for which focal infarcts of different sizes were induced in the posterior limb of the internal capsule. Immediately after the injection, flaccid paralysis was observed in the hand contralateral to the injected hemisphere. Thereafter, dexterous hand movements gradually recovered over months. After movement recovery, task-evoked hemodynamic responses increased in the ventral premotor cortex (PMv). The response in the PMv of the infarcted  hemisphere increased in the monkey that had received less damage. In contrast, the PMv of the non-infarcted  hemisphere was recruited in the monkey with more damage. A pharmacological inactivation experiment with muscimol suggested the involvement of these areas in dexterous hand movements during recovery. These results indicate that fNIRS can be used to evaluate brain activity changes crucial for functional recovery after brain damage. Compensatory activity changes in the brain areas that remain undamaged are thought to underlie functional recovery16. Therefore, it is important to monitor brain activity during rehabilitative training to determine whether the training is actually inducing appropriate brain activity changes. Clinical studies in human stroke patients, as well as experimental studies in animals in which damage is artificially induced in a specific region in the brain, have helped to elucidate the compensatory changes of brain activity that occur during the course of functional recovery17.Neuronal motor systems have the capacity for functional recovery following brain damage such as that induced by stroke, and functional recovery can be enhanced by postlesion rehabilitative training19. The motor cortex and corticospinal tract of macaque monkeys are more comparable to those of humans than are those of other animals used in experimental research (see ref. 20. for review). This motor system homology with that of humans, in combination with the relatively large macaque brain, makes imaging data obtained in macaque monkeys comparable to data obtained in clinical research. Therefore, studying macaque models of brain damage can facilitate the translation of findings to stroke patients.We previously established a macaque model for inducing artificial damage in brain regions involved in the transduction of motor commands215O-positron emission tomography (PET) scans in macaque monkeys, we previously showed that the ipsilesional ventral premotor cortex (PMv) is involved in the recovery of dexterous hand movements after lesioning of the primary motor cortex (M1)21. Enhanced involvement of the PMv was also observed with functional magnetic resonance imaging (fMRI) of stroke patients during recovery of hand movements14, indicating that this area is a candidate for assessing the effectiveness of rehabilitative training for hand motor dysfunction after brain damage. PET scanning is, however, not appropriate for monitoring brain activity in clinical rehabilitation because of the invasiveness of using radioactive tracers. Moreover, the subject needs to be fixed in the scanner during PET scanning and fMRI, and their natural posture and movements, such as those used during rehabilitative training, are therefore restricted. Functional near-infrared spectroscopy (fNIRS) offers potential for recording in unrestrained subjects, including those performing rehabilitative training, although it is essential to remove artifacts originating from body motions and optode fluctuations. Previous studies using fNIRS in stroke patients have reported compensatory changes of brain activity compared with that in normal subjects (see ref. 24. for review). Experimental studies using lesioned animal models will allow the brain activity changes caused by rehabilitative training to be evaluated more clearly, because brain activity after functional recovery can be compared with that measured before brain damage in the same subjects. Moreover, the causal role of the brain areas where activity changes are observed in functional recovery can be confirmed in animal models by the use of invasive pharmacological inactivation.Brain damage related to hand functionality can greatly affect the quality of human life and is therefore an important area of study. Using H25, we developed an fNIRS system for monitoring macaque cerebral motor activity during voluntary movements without head fixation. We recently used this system to successfully monitor functional hemodynamic responses in motor-related cortical areas reproducibly over long periods of time. Here, we used this system to investigate whether brain activity changes during functional recovery after brain damage could be evaluated by fNIRS.In a previous study18. The internal capsule carries the corticospinal tracts, and the severity and outcome of motor impairments depend on the degree of damage to this area. After the recovery of dexterous hand movements with postlesion motor training, the hemodynamic response evoked during a small-object retrieval task was measured again. In addition to the fNIRS measurements, another experiment was performed in which we pharmacologically inactivated the region where hemodynamic responses increased during functional recovery by using muscimol, a gamma-aminobutyric acid receptor agonist. We confirmed that local inactivation of the active regions identified with fNIRS measurement reversed the recovery of hand movements in the small-object retrieval task.We first measured the hemodynamic responses evoked during a small-object retrieval task before infarction Fig. . Focal i25; ΔHbO and ΔHbR exhibited opposite directional changes, and larger increases in ΔHbO and decreases in ΔHbR were recorded at channels in the hemispheres contralateral to the hand being used. As in the previous study, paired t-tests were conducted for each Hb species for each channel in each session to statistically evaluate the difference in Hb changes between use of the left and right hands  onto the anatomical MR image for each monkey indicated that channels with highly significant t-values in ΔHbO were distinctively localized around the hand area of M1 . The hemodynamic responses were similar between the two monkeys and consistent with our previous reportnds Fig. . Color-m M1 Fig. .Figure 2Before endothelin-1 injection, monkeys performed the Klüver board and vertical slit tasks smoothly with dexterous hand movements that included a precision grip, holding the morsel between the tips of the index finger and thumb (Supplementary Video S1). Immediately after endothelin-1 injection into the posterior internal capsule of the left hemisphere, motor paralysis was observed in the contralateral (right) forelimb. The macaques showed impaired performance in the small-object retrieval tasks and no precision grip in the right hand during the first month after the injection.18. The enduring brain damage was also confirmed histologically in Nissl-stained brain sections after perfusion fixation . Although hemodynamic responses were evoked by hand movements during the small-object-retrieval task after recovery, their spatiotemporal patterns were different from those before infarction and, moreover, differed between the two monkeys. In Monkey 1, the hemodynamic response was enhanced not only in the M1 hand area but also in cortical areas located rostrally to M1 in the ipsilesional (left) hemisphere, including the PMv Fig. . By contWe applied two-way MANOVA to find interaction effects between infarction (pre/post) and the hand used (left/right). In Monkey 1, statistically significant F-values were found in ipsilesional (left) hemisphere channels 20, 21, 22, and 26, which corresponded to M1; channels 1,7, 8, and 16, which corresponded to the PMv; and channels 9 and 17, which corresponded to the dorsal premotor cortex (PMd) Fig. . Among tt-values in the PMv areas was slightly earlier than that in the hand area of M1 (channels 21 and 22 of Monkey 1 and channels 17 and 18 of Monkey 2), but the difference was not statistically significant ; it was also significantly earlier than that in M1 after recovery .In both monkeys, the peak time of the hemodynamic responses in the PMv areas—channels 8 and 16 of Monkey 1 and channel 11 of Monkey 2  PMv where the most prominent enhancement in activation was observed. The muscimol injections did not have a significant effect on performance on the Klüver board task when the well diameter was 20 mm Fig. , but it 21. The involvement of PMv during functional recovery of hand movements has been reported in other studies of brain-damaged monkeys27 and in stroke patients14. Our current results, in which activation was enhanced more in the ipsilesional PMv of the monkey with less damage (Monkey 1) and in the contralesional PMv of the monkey with more damage (Monkey 2), are also consistent with data from stroke patients and brain-damaged animals. Previous studies have indicated that functional reorganization of the motor cortex may occur in both the ipsilesional and contralesional hemispheres during functional recovery after stroke, and that the contralesional motor cortex may play a greater role in recovery when damage is more severe, because neuronal reorganization for functional recovery within the ipsilesional side may be difficult to accomplish under these circumstances28. In our previous PET study using M1-lesioned monkeys, enhanced activation was found in the ipsilesional PMv21; by 2 months after lesioning, the success rate of precision grip in the M1-lesioned monkeys had recovered to within the 95% confidence level for performance before the injection. This shorter period required for performance recovery than the 6 months needed for Monkey 1 indicates that the M1-lesioned monkeys in our previous study had less-severe brain damage than Monkey 1 in our present study. Involvement of the ipsilesional PMv has been reported by other studies, in which a partial lesion was made in the M1 of monkeys27. Therefore, the present results for Monkey 1 are consistent with those of the previous studies:27 activation of the ipsilesional PMv was enhanced during recovery of hand movement after mild brain damage. Further analysis with additional monkeys is needed to elucidate the factors that determine the hemisphere involved in functional recovery after unilateral brain damage.Using fNIRS allowed us to demonstrate robust changes in the task-evoked hemodynamic response after infarction. After the recovery of hand movements, task-evoked hemodynamic responses increased in areas within the PMv. This result is consistent with our previous study using PET scans in macaque monkeys, which showed that the PMv is involved in the recovery of hand movements after lesioning of M130. However, the hemodynamic responses showed much greater enhancement in the PMv than in M1 and the PMd, possibly because the PMv is involved in grasping and manipulating objects32, so that the small-object retrieval task specifically activates this region. It will be important to address the contributions of M1 and the PMd to further elucidate the mechanisms underlying motor recovery after infarction in the posterior internal capsule. Moreover, the supplementary motor cortex (SMA) plays a role in motor recovery after brain damage36. However, our two-way MANOVA analysis did not indicate very notable differences between the pre-lesion and post-recovery conditions in the channels above the SMA ; further analysis with a larger sample size is needed to determine how the changes in response timing are related to the size of the infarcts. Despite the small sample size, we were able to detect such changes in response timing because fNIRS has greater time resolution than fMRI and PET. Another limitation was that the functional assessment with fNIRS and the reversible inactivation were performed at a single time point during the plateau phase of motor recovery. It is possible that different areas may play a role at other time points—in particular during the earlier phase preceding the plateau, as suggested in previous brain imaging studies25, in which the hemodynamic response in the intact motor-related cortical areas was measured with the fNIRS system described below. The animal-use protocol was approved by the Institutional Animal Care and Use Committee of the National Institute of Advanced Industrial Science and Technology (AIST) and was implemented in accordance with the “Guide for the Care and Use of Laboratory Animals” . The monkeys were housed as described in our previous study21; they were housed in adjoining individual primate cages  that allowed social interaction under controlled conditions of humidity, temperature, and light; they were monitored daily by the researchers and animal care staff to ensure their health and welfare. The housing area was maintained on a 12-h light/12-h dark cycle, and all experiments were conducted during the light cycle.Two Japanese macaque monkeys were used Table . The mon25, a high-spatial-density fNIRS measurement system was developed for monitoring cerebral functional hemodynamic responses in monkeys. Its measurement reproducibility was confirmed in observations of functional hemodynamic responses in motor-related cortical areas of Monkey 1 in the intact condition. There, fNIRS optodes were directly affixed to the skull surface of the macaque both to ensure accurate absolute spatial placement of the optodes and to eliminate physiological signals originating from sources other than the brain. The possibility of signal contamination from tissues other than the scalp was also considered. Diploic veins are present in the skull itself; however, it is still unknown whether the blood flow in these veins changes upon task execution. Our previous study using the same experimental setup as in the present study revealed a significantly high correlation between ∆HbO and ∆HbR in almost all channels25. This strongly suggests that the hemodynamic response measured by our present system is caused by neurovascular coupling, because other hemodynamics—including those in the diploic veins—would reflect neither regional oxygen consumption nor regional washout of HbR by overcompensation of HbO. We applied the hemodynamic modality separation method46, which separates the fNIRS signal into a cerebral functional component and a systemic physiological component, to our experimental data. The separated systemic physiological component in each channel was very small and was not correlated with its original fNIRS signal (data not shown). On the basis of these findings, we conclude that signal contamination from blood in the diploic veins was negligible in the fNIRS data that we obtained.In our previous study25. Briefly, the positions of the central and arcuate sulci were determined by using stereotaxic coordinates from T1- and T2-weighted MR images of each monkey’s head, obtained with a 3.0-T MRI system   were obtained to identify the position of each optode. The optical partial path length in the cortical layer at every measurement channel was calculated with a computer simulation of light propagation in monkey head models containing the sophisticated anatomical structure from both the T1 and T2 MR images. The optodes were fixed into the socket wells just before every experimental session. To detect fNIRS signals with a high spatial density of several millimeters in the inter-channel interval, a triangular bidirectional measurement was implemented, as described in our previous study25.The procedure used in the present study was almost identical to that of the previous studyds) Fig. . This ands) Fig. . Under p47. In this task, the monkey sat in a primate chair and retrieved a small, spherical food pellet (5 mm in diameter) from the Klüver board, which contained cylindrical wells of three different sizes . The size of the well was fixed within a daily session, and the monkey retrieved food pellets approximately every 20 s by using each hand alternately. One hundred fifty trials (75 for each hand) were performed in each daily session. The onset of a food-retrieval movement by either the left or right hand was detected by a digital laser sensor , which was fixed at the slit in front of the food well. The onset was defined as the time point when the sensor detected the monkey’s upper limb. From the recorded video data, we confirmed that the period of time between this onset and the start of digital flexion differed among most trials within only several tens of milliseconds.The fNIRS measurements were obtained as the monkeys performed a small-object retrieval task, the Klüver board task Fig. , closely18. A craniotomy was made over the internal capsule, and endothelin-1 —a vasoconstrictor peptide48—was then injected into the internal capsule via a microsyringe . To evaluate the size of the infarcts in each monkey, MRI scans were performed 1 month after the injection; the area of infarcts was seen as a hypointense core with a surrounding hyperintense rim on T2-weighted MR images. The unbiased volumes of the infarcts were calculated on the basis of Cavalieri’s principle49 by using StereoInvestigator imaging software .Focal infarcts were induced in the posterior limb of the internal capsule by using the same procedure as in our previous work47. In the test session with this task, the macaques retrieved a small piece of sweet potato (7 × 7 × 7 mm in size) attached to a needle tip in a cylindrical tube (20 mm in diameter), for 20 trials. The cylindrical tube was located at shoulder height and at a sagittal distance of 150 mm from the cage. Successful retrieval was defined as the monkey retrieving the sweet potato and bringing it to its mouth within 30 s and without dropping it. The proportion of successful retrievals was determined.Performance of hand movements before and after the endothelin-1 injections was evaluated by means of the vertical slit task, a small-object-retrieval task performed in the primate cage and identical to that used in our previous studies50.The region where hemodynamic responses increased most during functional recovery after the infarcts was inactivated by using muscimol and a procedure similar to that used in our previous studiesThe sites of muscimol injection were determined from the results of the fNIRS measurement. For Monkey 1, muscimol was injected into the center of two measurement channels in which the hemodynamic response evoked by the small-object-retrieval task dramatically increased after the recovery of hand movements. The effects of muscimol injection on hand performance were evaluated by using the Klüver board task. The test session to evaluate hand performance was conducted between 40 and 60 min after the muscimol injection. It consisted of 20 trials for each well size  for each hand, i.e., 120 trials in total. For Monkey 2, the measurement channel in which the hemodynamic response increased most dramatically after the recovery was located in the PMv in the hemisphere contralateral to the infarcts. We injected muscimol into this location and also into the corresponding location in the ipsilateral hemisphere, and we then compared the effects of inactivation of the PMv in each hemisphere. Unfortunately, Monkey 2 did not perform the Klüver board task in a sitting position, probably because it was more tired of sitting in the primate chair for a long period under the effect of muscimol. Indeed, this monkey was able to perform small-object retrieval when it was returned to its cage. Therefore, we evaluated the effect of inactivation with the vertical slit task. Twenty trials were conducted for each hand in the 40 to 60 min after muscimol injection. For both monkeys, an equivalent volume of vehicle (0.1 M phosphate buffer at pH 7.4) was also injected into the same injection sites. The muscimol and vehicle injections were performed three times with intervals of at least 2 days, because the effects of muscimol can persist until the day after injection.50. Tissue preparation was performed as previously described54. Images of Nissl-stained sections were acquired with a microscope  equipped with a 3CCD color video camera  and digitized with an image analysis system .The brain damage caused by the infarcts and the locations of the muscimol injections were confirmed histologically in Nissl-stained brain sections as in our previous studiest-test between the left-hand trials and right-hand trials, as in our previous study25. The differences in the ∆HbO and ∆HbR values between the fNIRS data sets of the left-hand trials and right-hand trials before and after infarction were analyzed by two-way multivariate analysis of variance (two-way MANOVA). Total Hb is also an important index corresponding to the regional cerebral blood volume; however, its change is smaller and less responsive to task execution than ∆HbO and ∆HbR, because HbO and HbR in the functional response usually change in opposite directions to each other. Therefore, for purposes of simplification, we did not focus on the total Hb change. The statistical significance of the difference in peak times of hemodynamic response between the PMv and M1, as well as between each time point, was assessed with the Tukey–Kramer test after one-way ANOVA, and that in the behavioral changes after muscimol injection was assessed with the two-sided Fisher’s exact test. All statistical analyses were performed by using the programming language R . R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org.)55 with Rstudio software . Tiled graphs of the time course for each statistical analysis were created with the software Igor Pro . The calculation of simulated light propagation and creation of the spatial activation maps overlapping with anatomical images were performed with Matlab .The fNIRS data from the 150 trials  were time-locked (t = 0) to the onset of food retrieval movement for that trial. Then the values of ∆HbO and ∆HbR for each time point for each channel for each monkey were statistically analyzed by using a paired Supplementary Information.


During the first wave of the COVID-19 pandemic, industries and academic institutes have collaborated to resolve the worldwide medical supply shortage issues. Innovative designs of 3D-printed items were proposed and developed by the maker community as a temporary solution to address the lack of personal protective equipment. An overview of global ongoing and past initiatives during the COVID-19 pandemic along with their challenges on retrofitting full-face snorkeling masks for healthcare applications such as splash-proof face shields, respirator masks and non-invasive ventilation systems are reported in this contribution. This study reviews these global initiatives and challenges. From our analysis, the present situation highlights the need to build solid networks between healthcare institutes and the different rapid prototyping initiatives. A clear feedback system needs to be implemented to facilitate effective collaboration between engineering (maker) and healthcare teams, to optimize the available human resources, and to achieve adequate product developments responding to the needs of healthcare workers. Viral diseases have been the cause of serious regional infectious outbreaks and pandemics in the last fifty years . The curOverall, the socioeconomic burden of the imposed travel and trade restrictions due to the current pandemic cannot be underestimated. It was projected that by the end of 2020, the impact solely due to aviation losses might negatively reduce the World Gross Domestic Product up to 1.6%, while job losses may rise to the value of 25–30 million .Decay of stock supplies ,13,14;Monopoly of the medical supply production  ,16;Limitations of commercial exchanges caused by transport regulations ,18;Increase of product prices due to greater demand ,19;Lack of flexibility and agility of product supply chains in times of crisis ,21,22.To slow down the SARS-CoV-2 transmission rate, governments and the World Health Organization (WHO) employed several measures such as personal hand cleaning, application of face masks and social distancing . The extIn this context, different initiatives from industry, academic institutes and the community strongly contributed to resolve this medical supply shortage issue . SeveralThe current pandemic situation revealed the need to build solid networks between “makers”  and “users”  for effective production of relevant medical supplies meeting the demand for HCWs . Among tThe aim of this investigation is to review the global initiatives on the use of snorkeling masks during the COVID-19 pandemic. Full-face snorkeling masks have been used to ensure physical or biological protection against the spread of the virus . In addiAlthough many initiatives were reported online, there is a lack of comprehensive data related to design and fabrication details as well as geographical location of the developed initiatives on retrofitting full-face snorkeling masks. In this communication, we aim to address this issue by providing an overview of ongoing and past initiatives along with their challenges on retrofitting full-face snorkeling masks for healthcare applications during the COVID-19 pandemic. This contribution concentrates on reviewing developed “connectors”  to repurpose full-face snorkeling masks for healthcare applications, such as splash-proof face shields, respirator masks and NIV systems . This stDiscussion and presented on a worldmap grouped by their geographical location. It should be noted that these initiatives are mainly located in North America and Europe as they host the largest snorkeling mask companies.An initial search in online databases  was conducted between March and October 2020, to identify academic literature detailing the use and modifications of snorkeling masks during the COVID-19 pandemic. Additional searches followed until early December 2020. The terms “snorkeling or snorkel mask and diving mask” were used in combination with the terms “COVID” and “3Dprinting” during the searches in these databases. Additional criteria  were also set on a daily basis to compensate the limited sources available. However, it must be noted that a large part of the developments and initiatives could be found only on websites, as most initiatives focused on rapid dissemination of their findings and results. For this reason, this review combined scientific articles as well as online data retrieved from reliable public agencies or institutions. All articles and information were rigorously verified on relevance and quality before downloading and use as an information source. Considering the rapid evolution of the COVID-19 pandemic, it is important to note that many documents are rapidly published, and information was updated on a daily basis. The present work reports the innovative designs of 3D-printed items, which were adapted to existing non-medical masks . InsteadThe proposed user strategies for using snorkeling masks in health care are summarized in In the first case a, the maIn the second case b, the snSubea Easybreath ;Seac Unica ;Siropack C-Voice, ;Ocean Reef Aria QR+ .The first adapter was for general use, where a sterile environment was not required. In this case, the one-way silicone valve , present near the mouth/chin area, allows exhausted air to be expelled from the lower part of the mask. This design corresponds more to a respirator mask with a breather valve, which protects the user but does not filter its exhalations . The sec® designed mask connectors integrating standard commercial filters  . T. T® desil grade) . Howeverl grade) ). Previol grade) , such asl grade) : when all grade) . Anotherl grade) . Based ol grade) , the modIn the third case c, standa® from Flow-Meter S.p.A). Some authors also reported the possibility to connect the system directly to the oxygen source without any flowmeter [2) during NIV [2 that can be obtained via commercial Venturi valves is usually limited to 60%. In order to increase the FiO2, a modified device using a Venturi valve with a secondary inlet was suggested [In a conventional ventilation method, the standard medical mask is connected directly to a CPAP module . This situation of direct connection of a mask to a respirator/ventilator device is outlined in lowmeter , having ring NIV . Howeveruggested , as show2O, 22 m, Intersurgical) was also integrated to control the positive pressure inside the mask. To limit the spread of the virus outside the closed circuit, two breathing filters  were positioned at the entrance and exit of the circuit. Some of the MMs used a reservoir bag placed in the inlet  to increase the air volume and pressure during the first seconds of inspiration. A one-way directional valve  was often placed between the mask and the entrance of the gas flow to reduce the dead space. This one-way directional valve is placed after the bag reservoir to decrease any CO2 accumulation during the respiratory cycle. A disposable manometer  could also be connected to the circuit to measure the internal pressure for improved system control. Different commercial adaptors were adopted  to fit the different items together. Actual case studies conducted on virus-infected patients are scarce in literature. Wagner et al. [Most of the MM designs used commercial medical tubes  to connect the mask to an available gas supply. An adjustable positive end-expiratory pressure (PEEP) valve . The geographical origin (including references) of the main snorkeling mask healthcare retrofitting initiatives over the past months have been highlighted in Stock availability/price of commercial masks,Availability and price of the commercial product computer-aided design (CAD) files,Designs covered by copyright or patent,Access to dimensional measurement tools.Despite the geographical distribution of these collaborative works, it remains important to note that no cost/benefit analysis has been done yet. The capacity to revert the production of standard engineering-related commercial products into medical devices  is a time-consuming process which can potentially lead to geometrical/dimensional errors, resulting in lower product performance . Hence, the following aspects should be considered when producing high quality and safe 3D-printed products:It is also important to note that among the different 3D printing technologies, Fused Deposition Modeling (FDM) has been largely used during the pandemic . This 3D(1)2 retention low to ensure safety after the adaptations?Is the CO(2)Is the protection sufficient against the viral load of SARS-CoV-2?(3)Is the pressure during the expiration and the expiration safe in NIV devices?When the first COVID-19 pandemic wave took place, many manufacturers declared on their websites that those devices were not subjected to a medical peer review and therefore not yet medically certified. Currently, minimal data support the use of uncertified medical devices in healthcare institutions. To our knowledge, an unofficial list  was reported by Isinnova et al. . Up to n2 retention inside the mask has no significant impact on arterial blood gas parameters during the test [2) and carbon dioxide (PCO2) values dissolved in the blood on a modified Ocean Reef’s mask during surgical procedures [2 were within normal ranges and the parameters indicate correct breathing and normal air circulation in the mask.In this context, Tack et al. concluded that COthe test . Neverthocedures . The PCO2 (FiCO2) for the full-face snorkel mask values. Their CO2 levels were higher than 1% after 30 min, which is above the health and safety regulations for respiratory PPE [2 FIO2, end-tidal oxygen (ETO2) and end-tidal CO2 (ETCO2) showed no changes), the modified mask failed other quantitative tests (this work has been limited to only a single user and a single combination of adaptor and mask). Germonpre et al. reported that a small initial increase of CO2 is observed at the end of an exhaled breath . However, this increase remains within the physiological limits and the authors conclude that 3D-printed adaptors used with commercial snorkeling masks  are safer, have more flexibility and reliability than makeshift adaptations [However, contradictory studies are published concerning the suitability to use these types of MMs in the medical field. To date, this results in a lack of confidence in using these MMs from HCWs . For exatory PPE . In additory PPE . Even ifptations .It is also important to note that most deployed filters have a bacterial and viral filtration efficiency of 99.999%. While different configurations of filtration exist , all filters for ventilatory use are equally effective . A revie2 and a positive pressure delivery system. During the experiments, the snorkel mask delivered CPAP levels up to 14 cmH2O. However, while CPAP masks have a small decrease in FIO2 (0.8%/cmH2O) with the increased pressure, the snorkel mask demonstrated stronger decline in FIO2. This particularly occurred when CPAP >12 cmH2O and this situation corresponded with an increase in leaks. This is particularly problematic in the context of a pandemic as aerosolization of the virus will then occur. In this work, the snorkel mask also demonstrated a higher CO2 accumulation  compared to the CPAP mask . Similarly, Alberto Noto analyzed the efficiency of an EasyBreath® mask as an emergency respiratory interface for a CPAP system [2) rebreathing was also recorded during spontaneously and resistively loaded breathing. Their experiments were made on 10 healthy volunteers with a high-flow system generator. The authors report substantial CO2 rebreathing for flow rates below 80 L min−1. The highest inspired CO2 partial pressure (PiCO2) was recorded at 40 L min−1 (min flow rate) with CPAP 15 cmH20 (max CPAP level). This was mainly related to the wide dead space (~900 mL) inside the mask. The use of a turbine-driven ventilator or high flows are necessary to wash-out the exhaled CO2 completely. Similarly, end-inspiratory to end-expiratory pressure swing, which is related to respiratory effort, was clinically relevant (>3 cmH20) only with a flow rate lower that 80 L min−1. The use of a reservoir bag or a threshold PEEP valve showed no improvement. To date, there is no study which reported a complete analysis obtained with unhealthy  patients. This implies that additional tests, made with a more statistical and medical methodology, seem necessary to highlight the relevance and the safety of this approach in a medical context not impacted by a temporal shortage of commercial products.It should also be noted that only a few studies have been reported in the literature concerning the performance of the MM for alveolar recruitment as well as gas exchange when used as a CPAP device. In this context, Landry et al. compared the performances of a snorkel mask and oronasal CPAP on two healthy male volunteers . The expP system . The autAssistance publique – Hôpitaux de Paris (AP-HP), among the largest hospital systems in the world, created the largest 3D print platform in France  of medical supplies is to ensure direct collaboration between designers and HCWs with constant feedback and adequate testing. Arash et al. described a research protocol for rapid iteration of reusable face shield design coupled with clinical feedback and real-world testing . The autprinters ) in collprinters  and a 3Dprinters . The hosprinters  to suppoprinters : mass prprinters , ensurinprinters  for the printers  and Mareprinters . The CADprinters , which mprinters , Parianiprinters , FPZ [13printers , PIUSI [printers , Campeteprinters , Ferrariprinters , Everex printers , SB3 [13printers  and Innoprinters ) joined printers ,138,139.printers .−1, 80 L or with a high-performance turbine-driven ventilator”.In conclusion, the integration of AM/3D printing for new medical device production is a flexible and attractive solution to counteract the needs of HCWs and patients during a pandemic. Small-scale three-dimensional (3D) design and printing laboratories have been widely used during the pandemic to increase the local production by parallelizing processes and sharing their resources. As highlighted in this work, many global initiatives have been supported by the maker and academic communities. Specifically, retrofitted snorkeling mask for usage as PPE or oxygen respiratory devices has been possible thanks to the development of 3D-printed connectors . Different 3D designs, prototypes and knowledge of retrofitting full-face snorkeling masks have been shared online during the pandemic. From a technical point of view, different parameters should be considered to ensure the advantage of employing 3D-printed products: (1) The final cost should be reasonable to guarantee the manufacture at large scale; (2) geographical availability of consumable materials and printers should be optimized as hospitals have often centralized supply demand; and (3) the final printed product must demonstrate a medical efficiency comparable to commercial items with similar features. Several concerns on the quality of the manufactured products (connectors) were raised when using the AM method and care should be taken in using the conditions to realize best connector–mask connections. To date, the safety of most proposed innovative designs remains under examination. For this reason, collaboration between researchers and medical industries along with proper standardization of new medical devices are prerequisites to establish “living lab” approaches. A proper and structured approach must be followed by all the involved parties, both on the engineering manufacturing side and the medical teams. A clear feedback system also must be implemented, ideally with a small ‘in situ’ lab in the medical institution, facilitating the collaboration between the involved teams and optimizing the available human resources. This is particularly true for the use of MM as NIV devices. Indeed, as highlighted by Noto et al. : “The moThe presented study provides a comprehensive review on ongoing and past initiatives on development of urgently needed PPE and other healthcare-related products during the first wave of the COVID-19 pandemic, to facilitate knowledge dissemination aiming to accelerate effective use of rapid prototyping in the context of pandemics.


This study aimed to evaluate high fat medium (HFM) effect on the gene expression profile of human Sk-hep1 cells and to determine critical differential proteins.There is a correlation between high fat diet (HFD), obesity, and non-alcoholic fatty liver disease. Despite wide range of investigations, understanding molecular mechanism of HFD effect on onset and progression of NAFLD warrants further examination. In this study, network analysis is applied to obtain a clear perspective about HFD effects and NAFLD.Gene expression profiles of human Sk-hep1 cells treated with HFM versus controls were extracted from GEO. Data were analyzed by GEO2R where the significant and characterized DEGs were included in the PPI network. The top 10 nodes of query DEGs based on four centrality parameters were selected to determine central nodes. The common hub nodes with at least other one central group were identified as central nodes. Action map was provided for the introduced central nodes. Heterogeneous nuclear ribonucleoprotein family including A1, A2/B1, D, R, and D-like, and five proteins  were introduced as differential proteins. mRNA processing and several biological terms including hypoxia and oxidative stress, apoptosis, regulation of cell morphology and cytoskeletal organization, and differentiation of micro tubes were introduced as dysregulated terms under HFM condition. In this evaluation, the role of high fat diet is confirmed in progression of NAFLD  first coined by Ludwig et al at 1980, is a serious liver disease caused by progression of non-alcoholic fatty liver disease (NFLD). NASH is associated with obesity and is characterized by liver inflammation, liver steatosis, and metabolomics syndrome . SeveralRecently network analysis of diseases has attracted the attention of many scientists. In this approach, large numbers of genes or gene expression products which discriminate patients from controls interact to construct a network. In scale free networks, there are several elements that play function as crucial components in the interactome. These critical elements are useful tools to find the mechanism of diseases or those that may be responsible for onset and progression of diseases , 11. PreSix gene expression profiles of human Sk-hep1 cells including three samples treated with high-fat medium versus three samples as growth medium (GM)-treated cells were extracted from GEO. The samples are introduced as GSE109836 and GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array. The incubation time was 12 hours and total RNAs from the two groups were extracted for the entire transcriptomic analysis. Data were analyzed by GEO2R and the top 250 DEGs were determined. Considering Fold change 2  and P-value ≤ 0.05, the characterized DEGs were identified and included in the PPI network. Among the various isoforms, the DEGs with the maximum value of expression change were selected. The DEGs were included in the PPI network via STRING database by Cytoscape software. Due to poor interaction between the nodes of the constructed network, 100 neighbor genes from STRING database were added to the query DEGs to construct a scale free network. Top 10 nodes of query DEGs based on four centrality parameters; degree (D), betweenness centrality (BC), closeness centrality (CC), and stress (S) were selected to determine the central nodes. The common hub nodes with at least one other central group were identified as the central nodes. The connections between the central nodes were determined via a sub-network. The action map including binding, expression, activation, and inhibition was provided for the introduced central nodes. After evaluation, the potent central nodes were identified and introduced as possible biomarkers related to the effect of high fat nutrition on the treated cells. Boxplot analysis is a suitable method to validate the possible comparability between samples. Since the distribution of data is median center see , the genThe connection between 10 central nodes is shown in the illustrated sub-network in The gene expression profiles of high fat treated samples were comparable with controls based on the distribution of gene expression criterion with the differentially expressed genes included in a scale free network. A total of 10 central genes affected by high fat treatment were identified. Five genes among 10 central genes are different kinds of heterogeneous nuclear ribonucleoprotein family including A1, A2/B1, D, R, and D-like sub families. Heterogeneous nuclear ribonucleoprotein family are involved in management of resulted mRNAs from transcription. As with the introduced five heterogeneous nuclear ribonucleoproteins, the other genes are almost related to the processing of mRNAs. The fundamental cellular processes such as cell cycle are associated to the function of the introduced central nodes see . Except It is reported that PRPF40A expression as with COL1A1and UCP2 is involved in the biochemical pathways that are related to the hypoxia and oxidative stress. This finding is providing by Urszula Oleksiewicz et al based on an investigation on the non-small cell lung cancer . Up-reguSRSF1 is the other central gene which is presented in PCF11 is the other protein introduced as a central protein. This protein is similar to many proteins that are involved in pre-mRNA 3′-end processing and transcription termination . LSM8 is the other critical protein that interacts with the rest of critical proteins except HNRNPDL (see table including LSM3-LSM8 which are involved in cis- and trans-splicing of mRNA .HSP90AA1 gene encodes heat shock protein 90α  which haThe literature findings suggest that the central nodes play a critical role in the promotion of high fat medium effects in the body. It seems that further investigation can be useful to introduce valuable biomarkers for diagnosis of disease and follow up of patients with fatty liver disorder. The analysis revealed that the heterogeneous nuclear ribonucleoprotein family and mRNA processing are the crucial proteins and biochemical pathways which are involved in the onset and progression of fatty liver disease. Several biological terms including hypoxia and oxidative stress, apoptosis, regulation of cell morphology and cytoskeletal organization, and differentiation of micro tubes were highlighted as processes dysregulated under high fat diet condition. The findings can suggest the discovery of a possible biomarker associated with the fatty liver disorder.


Participants rated attributes of characters who were described as being employed, working poor, unemployed or receiving unemployment benefits. The results show that compared to employed characters, unemployed characters were rated substantially less favorably at both time points on their employability and personality traits. The difference in perceptions of the employed and unemployed was, however, attenuated during COVID19 with benefit recipients perceived as more employable and more Conscientious than pre-pandemic. These results add to knowledge about the determinants of welfare stigma highlighting the impact of the global economic and health crisis on perception of others.Unemployed benefit recipients are stigmatized and generally perceived negatively in terms of their personality characteristics and employability. The COVID19 economic shock led to rapid public policy responses across the globe to lessen the impact of mass unemployment, potentially shifting community perceptions of individuals who are out of work and rely on government income support. We used a repeated cross-sections design to study change in stigma tied to unemployment and benefit receipt in a pre-existing pre-COVID19 sample ( Over just one month, from March to April 2020 unemployment rate in the United States increased from 4.4% to over 14.7% and in Australia the effective rate of unemployment increased from 5.4 to 11.7%  to enable employees to keep their employees connected to the workforce, one-off payments to many welfare recipients, and a doubling of the usual rate of the unemployment benefits (Jobseeker payment) through a new Coronavirus supplement payment. At the time of writing in July 2020, many countries, including Australia remain in the depths of a health and economic crisis.The onset of COVID19 pandemic saw unemployment climb to the highest rate since the Great Depression in many regions globallyto 11.7% 2. In AusA rich research literature from a range of disciplines has documented the pervasive negative community views toward those who are unemployed and receiving unemployment benefits, with the extent of this “welfare stigma” being particularly pronounced in countries with highly targeted benefit systems such as the United States and Australia . The stiNegative community attitudes and perceptions of benefit recipients are commonly explained by the concept of “deservingness” . The uneA number of experimental paradigms have been used to investigate perceptions of “welfare recipients” and the “unemployed.” The stereotype content model SCM; , for exaThe term “employability” has been previously defined as a set of achievements, skills and personal attributes that make a person more likely to gain employment and leading to success in their chosen career pathway , 2019. WBuilding on this previous work, our own research has focused on the effects of unemployment by drawing on frameworks of Big Five, SCM and employability in pre-COVID19 samples . Our stuEmployed; (2) Working poor; (3) Unemployed; and (4) Unemployed benefit recipient. Finally, for the COVID19 sample we added a novel fifth condition: (5) Unemployment benefit recipient also receiving the “Coronavirus” supplement. We except that the reference to a payment specifically applicable to the COVID19 context may lead to more favorable perceptions (more deserving) than the other unemployed and benefit receipt characters.The broad aim of the current study was to extend this previous work by examining the impact of COVID19 on person perceptions tied to employment and benefit recipient status. It repeats a pre-COVID19 study of an Australian general population sample in the COVID19 context, drawing on the same sampling frame, materials and study design to maximize comparability. The study design recognizes that the negative perceptions of benefit recipients may reflect a combination of difference sources of stigma: poverty, lack of work, and benefit receipt. Therefore, the original study used four different conditions to seek to differentiate these different sources: (1) The study capitalizes on a major exogenous event, the COVID19 crisis, which we hypothesize will alter perceptions of deservingness by fundamentally challenging social identities and perceptions of one’s own vulnerability to unemployment. The study tests three hypotheses, and in doing so makes an important empirical and theoretical contribution to understanding how deservingness influences person perception, and understanding of the potential “real world” barriers experienced by people seeking employment in the COVID19 context.3. We hypothesize that, at Time 1 (pre-COVID19 assessment) we will find that employed characters will be rated more favorably than characters described as unemployed and receiving unemployment benefits, particularly on dimensions of Conscientiousness, Worker and Boss suitability. Moreover, we expect a gradient in perceptions across the four experimental conditions, from employed to working poor, to unemployed to unemployed receiving benefits and to show a similar trend for the other outcome measures included in the study.The pre-COVID19 assessment uses a subset of data from a pre-registered study, but this reuse of the data was not preregisteredWe hypothesize that the character in the unemployed condition(s) would be rated less negatively relative to the employed condition(s) at Time 2, compared to Time 1. We predict a two-way interaction between time and condition for the key measures  and a similar trend on other outcomes.We expect that explicit reference to the unemployed benefit character receiving the “Coronavirus supplement” payment will increase the salience of the COVID19 context and lead to more positive ratings of this character relative to the standard unemployed benefit condition in the pre-COVID19 and COVID19 occasions.M Age = 42.1 [16.7] years, range: 18–82) participants who were presented with the one vignette scenario that we could replicate at the time of the social restrictions applicable in the COVID19 context . The sample for Time 2 (COVID19) was collected in May–June 2020, at the height of the lock down measures in Australia and included 670 participants . The two samples were broadly similar (see below), though the proportion of male participants at Time 2 was greater than at Time 1.Two general population samples (pre-COVID19 and COVID19) were recruited from the same source: The Australian Online Research Unit (ORU) panel. The ORU is an online survey platform that provides access to a cohort of members of the general public who are interested in contributing to research. The ORU randomly selects potential participants who meet study eligibility criteria, and provides the participant with an incentive for their participation. The sample for the Time 1 (pre-COVID19) occasion was part of a larger study (768 participants) collected in November 2018. From this initial dataset, we were able to use data from 260  participants then repeated this process with a second vignette, but we do not consider data from the second vignette.The key experimental conditions were operationalized by a single sentence embedded within the vignette that was randomly allocated to different participants . The four experimental conditions were identical at both time points. At Time 2, an additional COVID19-specific condition was included : “S/he is currently unemployed and is receiving government benefits, including the Coronavirus supplement, due to his/her unemployment.”All three study conditions will imply poverty/low income. In Australia, few minimum-wage jobs are supplemented by tips, and so a minimum-wage job indicates a level of relative poverty. A full-time worker in a minimum wage job is in the bottom quartile of income earners . Prior tSeveral characteristics of the vignette character, including age and relationship status, were balanced across study participants. Age was specified as either 27 or 35 years, relationship status was either “single” or “lives with his/her partner.” The character’s gender was also varied and names were stereotypically White.vignette template construction is presented in For Time 1, manipulated characteristics yielded 32 unique vignettes, comprised of four key experimental conditions  × 2 ages × 2 genders × 2 relationship statuses. For Time 2, manipulated characteristics yielded 40 unique vignettes, comprised of five key experimental conditions  × 2 ages × 2 genders × 2 relationship statuses. The In both studies, participants were required to affirm consent after debriefing or had their data deleted. Participant comprehension of the vignettes was checked via three free-response comprehension questions about the character’s age and weekend activities. Participants who did not answer any questions correctly were not able to continue the study.Personality, employability (suitability as a worker or boss), communion and agency, cognitive and emotional moral judgments, and dehumanization were included as the study outcomes. While not all personality or character dimension measures can be considered as negative or positive, higher scores were used in the study to indicate more “favorable” perceptions by the participants of the characters.The Ten Item Personality Inventory was used to measure the Big Five  and adapWorker suitability) and “I think [Name] would be a good boss” (Boss suitability) were rated on the same scale as the personality measure. The order of these two items was randomized. Higher scores indicated better employability.Single item measures: “I think [Name] would be a good worker”  scales that are new to this study and the previously tested Openness to Experience, our previous research has demonstrated differences between the ratings of employed and unemployed characters on the included outcome measures . We unde2  = 7.04, p < 0.001) and employment : The gender distribution was more balanced at Time 1 with 49.8% of males, compared to 59.5% of males at Time 2. There was also a significant increase in unemployment with 20.9% of Time 1 participants out of work compared to 39.3% of the Time 2 participants. This was likely reflective of the employment rate nearly doubling in Australia during COVID19 crisis. Bivariate correlations showed significant positive correlations between all 14 outcomes (p’s < 0.001), except for Extraversion that was only positively correlated with Emotional Stability, boss suitability, warmth, assertiveness, and competence (p’s < 0.05).The two samples from Time 1 (pre-COVID19) and Time 2 (COVID19) were comparable on all demographic variables, except for gender (χb = −0.57). This difference in the ratings of employed and unemployed characters was reduced in the COVID19 assessment at Time 2, declining from 0.57 to 0.26, across all the outcome measures. The addition of the three-way interaction between Condition, Time and outcome measure significantly improved overall model fit, χ2 (52) = 482.94, p < 0.001, indicating the interaction between Condition and Time varied over measures.The results, both adjusted and unadjusted, from the initial overall multilevel model using a binary indicator of whether vignette characters were employed (those in the employed or working poor conditions) or unemployed (unemployed or welfare) and testing the interaction between vignette Condition and Time (pre-COVID19 vs COVID19) are presented in the p’s < 0.05).A series of separate regression models considering each outcome separately see  showed ab = 0.13, 95% CI ) compared to males. Testing the heterogeneity of these patterns across outcomes via the inclusion of a three-way interaction between vignette condition, occasion and measure significantly improved overall model fit, χ2 (104) = 533.40, p < 0.001, prompting analysis of each outcome separately.The next set of analyses consider the four separate vignette conditions, differentiating between the unemployed and unemployed benefit recipient conditions. The overall mixed-effects multilevel model incorporating the four distinct vignette conditions provided evidence of significant effects for Condition and Condition by Time in both adjusted and unadjusted models. The result for the adjusted model , averageThe separate linear regressions for each outcome measure  show thab = 0.26, 95% CI ) and the general unemployed benefit recipient condition at Time 2  were both rated more favorably in comparison to unemployed benefit recipients at Time 1. There was no difference between these two Time 2 benefit recipient groups . These results did not support hypothesis 3.The inclusion of the fifth COVID19-specific unemployment benefit condition did not generate more positive (or different) ratings than the standard unemployment benefit condition. Overall mixed-effects multilevel models, both adjusted and unadjusted, indicated that participants in the Coronavirus supplement condition  and Conscientiousness. The more favorable ratings of unemployed and benefit receiving characters at Time 2 is likely to reflect how the exogenous economic shock brought about by the COVID19 crisis challenged social identities and the stereotypes held of othersbenefits . We suggbenefits . It is iIn contrast to our findings on the key measures of employability and Conscientiousness, the previous and current research is less conclusive for the other outcome measures. The current study showed a broadly consistent gradient in the perception of employed and unemployed characters for all outcome measures apart from Openness and Extraversion. Findings on these other measures have been weaker and inconsistent across previous studies , and theThe main findings of the current study are consistent with previous experimental studies that show that the stereotypes of unemployed benefit recipients are malleable . These pPerceptions and stereotypes of benefit recipients can be reinforced  by government actions and policies. Structural stigma can be used as a policy tool to stigmatize benefit receipt as a strategy to reduce dependence on income support and encourage workforce participation . In the The current study took advantage of a natural experimental design and replicated a pre-COVID19 study during the COVID19 crisis. The study is limited by the relatively small sample size at Time 1, which was not designed for current purposes but part of another study. We were not able to include most of the participants from the original Time 1 study as most of the experimental conditions described activities that were illegal/inconsistent with recommend activity at the time of the COVID19 lockdown and social restriction measures. Finally, the data collection for the current study occurred very quickly after the initial and sudden COVID19 lockdowns and economic shock, which is both a strength and a limitation for the generalizability of the results. The pattern of results using the same sampling frame offers compelling support for our hypothesis that the shared economic shock and increase in unemployment attenuates stigmatizing community attitudes toward those who need to receive benefits. Our current conclusions would be further strengthened by a subsequent replication when the public health and economic crises stabilize, to test whether pre-COVID perceptions return.The current study provides novel information about impact of the COVID19 health and economic crisis, and the impact of the corresponding policy responses on community perceptions. This novel study shows how community perceptions of employment and benefit recipient status have been altered by the COVID19 pandemic. These results add to knowledge about the determinants of welfare stigma, particularly relating to employability, highlighting societal level contextual factors.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Melbourne University Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.AS led the review conceptualized by TS and PB. AS and PB conducted the analyses and wrote up the review. TS led the data collection, reviewed and edited the manuscript, and provided data management support. This manuscript is based on previous extensive work by TS and PB on stereotypes toward the unemployed and welfare benefit recipients. 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.