Influenza A viruses (IAV) are highly contagious pathogens causing dreadful losses to human and animal, around the globe. IAVs first interact with the host through epithelial cells, and the viral RNA containing a 5′-triphosphate group is thought to be the critical trigger for activation of effective innate immunity via pattern recognition receptors-dependent signaling pathways. These induced immune responses establish the antiviral state of the host for effective suppression of viral replication and enhancing viral clearance. However, IAVs have evolved a variety of mechanisms by which they can invade host cells, circumvent the host immune responses, and use the machineries of host cells to synthesize and transport their own components, which help them to establish a successful infection and replication. In this review, we will highlight the molecular mechanisms of how IAV infection stimulates the host innate immune system and strategies by which IAV evades host responses. Influenza A viruses (IAV) cause highly pathogenic respiratory problems in human and animals. It is the major cause of annual epidemics and occasional pandemics in humans, responsible for 3–5 million cases of severe clinical infections and 250,000 to 500,000 fatal cases every year throughout the world Stohr, . InfectiIn addition to typical seasonal infections, IAV can also undergo substantial changes (recombination/antigenic shift) that leave imprints of infection with little to no protective immunity and uplift mortality rate even among healthy young adults infected with IAV , of viral proteins hemagglutinin (HA) and neuraminidase (NA), protruded out from the envelope. These spikes appear as either rod-shaped (HA) or mushroom shaped (NA). The ratio of HA to NA generally varies from 4:1 to 5:1. The high density of HA is probable to enhance the chances for viral attachment. Additionally, matrix protein 2 (M2) is also enriched into the lipid envelope. The ratio between M2 and HA is usually about 1:10 to 1:100  and innate immune responses , invasion of alveolar dendritic cells and macrophages  are remained in the cytoplasm. PRR sensing IAV infection causes activation of IRF3/7 through phosphorylation of the C-terminus of IRF3/7 and dimerization that leads to the exposure of nuclear localization signal (NLS), which in turn results in nuclear translocation of IRF3/7. On the other hand, the inhibitor of NF-κB (IκB) retains the NF-κB in the cytoplasm. IκB undergoes ubiquitination and proteasomal degradation upon sensing of appropriate signals by PRR. Removal of IκB leads to nuclear translocation of NF-κB via its NLS  like SOCS1 and SOCS3 are found to be involved in negative regulation of JAK-STAT pathway  normally expressed in various human cells plays an important regulatory role in host innate immunity. Upon infection with IAV, ssRNA virus (SeV), dsRNA virus (MDRV) or DNA virus (HSV), down regulation of NRAV was observed. Further investigations revealed that NRAV is critically involved in regulation of IAV pathogenesis, as evidenced by considerably higher virulence, enhanced acute lung injury and consequently decreased survival rates of transgenic mice overexpressing the lncRNA NRAV when compared with wild type mice. Particularly, mRNA levels of some critical ISGs were substantially reduced in NRAV-overexpressing cells and transgenic (TG) mice infected with IAV, including IFIT2, IFIT3, IFITM3, OASL, and MxA. These studies suggest that NRAV is a key negative regulator of some ISGs during IAV infection in vitro and in vivo. Therefore, down regulation of NRAV in IAV-infected cells might be a host self-protection response to the virus infection, which may be critical for viral clearance  are an important class of regulatory RNAs in a variety of cellular processes. Recently, it has been shown that a human lncRNA named NRAV  cells are important cytotoxic and effector cells of innate immunity that serve as a crucial first-line defense against tumors and virus-infected cells. It has been previously shown that lysis of IAV-infected cells is mediated by the interaction between the NK receptors, NKp44 and PKp46, and the IAV hemagglutinin (HA) type 1 expressed by the infected cells  to mitigate the host innate immune response to influenza virus infection. NS1 is considered being the most important IFN-antagonist protein encoded by the influenza virus. This protein is highly expressed in the cytoplasm and the nucleus of infected cells where it is able to interact with different components involved in the IFN-mediated immune responses and thereby inhibits these responses   during the viral entry process  Fig. .Figure 2IPK from hsp40 and minimizes the antiviral state of cell (Sharma et al., Interestingly, influenza viruses not only use their own proteins to evade the innate immune system, but also take advantage of specific cellular regulators of immune system to further minimize the antiviral effects by host cells. Recently, it has been demonstrated that influenza virus-induced SOCS1 inhibits the phosphorylation of STAT1 at early stages of infection (Wei et al., In last decade, our knowledge on innate immune responses against influenza infections has expanded significantly. As a result, serious efforts were made to utilize the gained knowledge for designing better vaccines and accompanying control measures. While our understanding increased on the virus-induced innate immunity, it remains to better understand the dynamics and breadth of innate immunity in tissue, species, and host-specific manners. Although more viral proteins are being identified in the influenza viruses, their importance in the influenza pathogenesis is not clearly elucidated. In particular, little information is available about their role in viral escape from host innate immunity. Bridging these gaps will pave the ways not only for designing better vaccines but also for developing novel antiviral agents.


Pannexin 1 (Panx1) is a high-conductance, voltage-gated channel protein found in vertebrates. Panx1 is widely expressed in many organs and tissues, including sensory systems. In the eye, Panx1 is expressed in major divisions including the retina, lens and cornea. Panx1 is found in different neuronal and non-neuronal cell types. The channel is mechanosensitive and responds to changes in extracellular ATP, intracellular calcium, pH, or ROS/nitric oxide. Since Panx1 channels operate at the crossroad of major signaling pathways, physiological functions in important autocrine and paracrine feedback signaling mechanisms were hypothesized. This review starts with describing in depth the initial Panx1 expression and localization studies fostering functional studies that uncovered distinct roles in processing visual information in subsets of neurons in the rodent and fish retina. Panx1 is expressed along the entire anatomical axis from optical nerve to retina and cornea in glia, epithelial and endothelial cells as well as in neurons. The expression and diverse localizations throughout the eye points towards versatile functions of Panx1 in neuronal and non-neuronal cells, implicating Panx1 in the crosstalk between immune and neural cells, pressure related pathological conditions like glaucoma, wound repair or neuronal cell death caused by ischemia. Summarizing the literature on Panx1 in the eye highlights the diversity of emerging Panx1 channel functions in health and disease. In higher vertebrates, the Panx gene family consists of the three glycosylated integral membrane proteins Panx1, Panx2 and Panx3  genes , inner nuclear layer (INL), the outer nuclear layer (ONL) and to a lesser extent in the inner plexiform layer  and on non-invaginating cellular processes at the base of rod spherules, resembling the flat contact between rods and cone bipolar cells. The authors also confirmed the expression of Panx1 in HC using single cell RT-PCR, IHC and ultrastructural analysis and found Panx1 localized at the tips of HC dendrites invaginating in rod spherules. It is worth noting that only one HC type is present in the murine retina receiving cone input at the dendrites and rod input at the axon terminals . Kranz et al.  of Panx1 in the processing of visual information were investigated using Panx1z et al.  demonstr2+ and pH changes  to demonstrate whether Panx1 is part of a pathological cascade leading to ischemia-induced RGC loss , and abolished when AGA was combined with the Panx1 specific inhibitor probenecid, or when apyrase was added to catalyze the hydrolysis of ATP. Using the exocytosis inhibitor N-ethylmaleimide (NEM), the authors ruled that ATP is released via exocytosis. Using the purinergic P2 receptor antagonist reactive blue-2 and pertussis toxin (PTX), the authors showed that the increased Na+/K+-ATPase activity under hyposmotic stimulation is most probably mediated by G-protein coupled P2Y receptor activation. Thus, increased Na+/K+ATPase activity of the epithelium during a hyposmotic challenge was prevented when either ATP release or extracellular ATP accumulation was suppressed, or P2Y receptors were inhibited. This result correlated Panx and connexin activities with ATP release, most likely due to the channels’ mechanosensitivity  of Panx1 in the lens epithelium were investigated using the porcine lens model  benzoic acid physiological roles of Panx1 needs to overcome biological, technical and logistical challenges. A significant biological challenge derives from the complex interactions of Panx1 with major signaling pathways involving ionotropic and metabotropic receptors (Isakson and Thompson, 10Panx1 (Pelegrin and Surprenant, Pharmacological manipulation of Panx1 is a second challenge despite the significant advances made in recent years. Blocking peptides like in vivo. A challenge emerging from mouse models and KO cell lines is the question of compensatory upregulation of other Panxs or connexin hemichannels. Recently, Panx3 has been shown to be upregulated in the arterial walls and skin of Panx1 knock out mice (Lohman and Isakson, The availability of Panx1 KO mouse models has greatly contributed to the understanding of Panx1 functions Dissecting complex interactions and solving pharmacological shortcomings are of general concern. A specific concern for vision and eye research is choosing appropriate animal models. While vertebrate eyes share a general anatomy and physiology, visual capabilities are distinct, frequently reflecting functional adaptations to habitats and/or behaviors. Complementary models are needed to weight advantages over disadvantages of each species when investigating the roles of Panxs in the eye. Already distinct functional roles of Panx1 in the mouse and zebrafish retina have emerged, reflecting unique properties of the two species (Kranz et al., ex vivo and in vitro preparations suitable to address the emerging roles of Panx1 in a physiological or pathological context, and the potential implication of Panx1 in leading causes of blindness. There is no confirmed unifying theme to be highlighted yet. Instead, we wish to communicate that Panx1 channels are highly versatile channels. Functions are mediated by the distinct localization of Panx1 proteins in different cell types, multiple protein interactions, as well as post-translational modifications. Since Panx1 channels operate at the crossroad of major signaling pathways, it is tempting to speculate that long lasting changes to this channel have the potential to impact physiological functions by altering major signaling pathways. ATP release, as found in the humor outflow system or as a mediator of cross talk between neurons and microglia, has the potential to be the unifying molecular function, connecting biophysical properties of Panx1 channels to ATP mediated signaling. However, this generalization might not be fully true. Bipolar cells like CB3a cells do not release ATP in any known physiological or pathological context. Further, HC release ATP, but it is the pH shift evoked by the breakdown of ATP to adenosine that alters neuronal communication. Since insight into the relevant molecular, cellular and physiological roles of Panx1 in conditions as diverse as primary processing of visual information in the outer retina or IOP regulation is emerging, we expect soon advances in conceptual insight related to the different roles of Panx1 in this physiologically and clinically important sensory organ.Panx1 research in the eye has moved from early expression and localization studies to functional studies. This research is currently driven by the availability of knock out animal models, sophisticated The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The goal here is to describe our current understanding of heme metabolism and the deleterious effects of “free” heme on immunological processes, endothelial function, systemic inflammation, and various end-organ tissues , with particular attention paid to the role of hemopexin (HPX). Because heme toxicity is the impetus for much of the pathology in sepsis, sickle cell disease (SCD), and other hemolytic conditions, the biological importance and clinical relevance of HPX, the predominant heme binding protein, is reinforced. A perspective on the function of HPX and haptoglobin (Hp) is presented, updating how these two proteins and their respective receptors act simultaneously to protect the body in clinical conditions that entail hemolysis and/or systemic intravascular (IVH) inflammation. Evidence from longitudinal studies in patients supports that HPX plays a Hp-independent role in genetic and non-genetic hemolytic diseases without the need for global Hp depletion. Evidence also supports that HPX has an important role in the prognosis of complex illnesses characterized predominantly by the presence of hemolysis, such as SCD, sepsis, hemolytic-uremic syndrome, and conditions involving IVH and extravascular hemolysis (EVH), such as that generated by extracorporeal circulation during cardiopulmonary bypass (CPB) and from blood transfusions. We propose that quantitating the amounts of plasma heme, HPX, Hb-Hp, heme-HPX, and heme-albumin levels in various disease states may aid in the diagnosis and treatment of the above-mentioned conditions, which is crucial to developing targeted plasma protein supplementation  therapies for patients with heme toxicity due to HPX depletion. The low, essentially constant leakage of hemoglobin (Hb) from red blood cells (RBCs), termed trivial hemolysis, is well-tolerated; however, both intravascular (IVH) and extravascular (EVH) hemolysis induce severe inflammation and potential tissue dysfunction. This pathology is due to heme (iron-protoporphyrin IX) released from methemoglobin , with particular attention paid to the protective role of HPX. We propose that the model of a linear pathway for the development of heme toxicity with HPX as a backup system for Hp, i.e., acting only when Hp becomes depleted, does not represent many situations While HPX's role in protection against heme-mediated inflammation and oxidative damage is established, further studies are needed to confirm the Hp-independent activity of HPX in hemolysis in various clinical disease states in humans. We have therefore summarized and reviewed both historical and recent observations on the HPX and Hp systems in relevant human conditions and diseases. Finally, we propose future research avenues related to heme metabolism in human health and disease. These include quantitating the amounts of plasma heme, HPX, Hb-Hp, heme-HPX, and heme-albumin levels in various disease states to aid in the diagnosis and treatment of diseases prominently characterized by hemolysis and investigating the use of targeted plasma protein supplementation  therapies for patients with heme toxicity due to HPX depletion. Although not addressed here, α-1 microglobulin binds heme and free radicals protecting particularly EVH sites , other hemolytic conditions and also in atherosclerosis  in the immediate plasma (550 μl). Given that total body plasma Hp would be ~2.8 × 1019 molecules 4], as two αβ globin dimers each monomer with its own heme bound  rather than of Hb is preferred in humans in contrast to mice  from average of 60 (range 40–70) at 1–12 years to average of 77 (range 66–100) at 20–40 years but then declines at 50–70 years  load , which is an NO-induced shock with severe hypotension and vasoplegia disrupting major organ function leading to death. PHP has many anti-oxidant properties due to a covalent linkage to catalase and can scavenge NO, and it does not readily oxidize either to metHb derivatives that do not bind O2 or to ferryl Hb. It was anticipated to provide treatment in ischemia- reperfusion injury and hemorrhagic shock, where ROS such as superoxide and H2O2 and NO are known to be involved. Three patients treated with PHP  were followed daily for 2 weeks. Plasma Hp levels remained normal but HPX was essentially undetectable by immune-nephelometry. Free Hb (measured as PHP Hb with a higher molecular mass than Hb upon starch gel electrophoresis) was detected in the patients undergoing PHP infusions. The patient's Hp could bind Hb because Hb-Hp complexes formed after Hb addition to plasma samples collected several days after the infusion occurred. Throughout this period, total bilirubin, and heme-binding serum α1-microglobulin levels were normal. Ongoing heme catabolism was apparent with total bilirubin levels increased for several days after PHP infusion. One patient had very low HPX levels initially that eventually rose to low normal levels as their elevated serum creatinine decreased slightly over the second week; and hepatic function improved too as the initially abnormally high aspartate transaminase and alanine amino transferases tended to decline in the second week. However, lactate dehydrogenase (LDH) levels were extremely high initially, increasing over days 3–10 then declining by days 12 and 14. In another patient whose HPX hardly responded, the LDH levels remained abnormally high. This group of patients provide an example of heme clearance by HPX with an increased hemolysis index value but with normal Hp.In an attempt to minimize the toxic effects of blood substitutes, Hb molecules have been designed to: survive for a long time in the circulation; bind OHPX will be important in clearing myoglobin (Mb)-heme from injured muscle in crush injuries. Mb is expressed in cardiac myocyte and smooth muscle cells. Mb levels, normally 30–90 ng/ml, increase 5- to 10-fold (~500 ng/ml) after myocardial infarcts  is the phagocytosis of senescent, damaged or otherwise abnormal RBCs by macrophages in the spleen, liver, bone marrow, and reticulo-endothelial system. Random hemolysis—or the age-Most pathological hemolysis is EVH with the spleen playing a major role. EVH is stimulated after one or more blood transfusions in order to clear any RBCs that have deteriorated upon storage. In this case, EVH and IVH likely occur simultaneously, and, depending upon the quality of the stored blood and the number of transfusions, the hemolysis can expose cells and tissues to a significant amount of Hb, heme, and iron. Transfusions can be extensive, for example, car accident victims may require 100 pints of blood, and there is the potential for a daily transfusion need during chemotherapy and a lifetime need in SCDAfter engulfment of RBCs by macrophages, the heme from Hb leaves the endosomes and reaches the smooth endoplasmic reticulum (ER) for catabolism by the HOs 1 and 2 (HO1 and HO2) where each heme molecule stoichiometrically releases redox active ferrous iron and carbon monoxide (CO). Heme also moves to the nucleus for gene regulation in part via depression of Bach1-regulated genes, including HO1 and NADP(H):quinone oxido-reductase 1 (NQO1). These molecules protect cells against oxidative stress as levels of redox active heme and iron rise intra-cellularly. The iron from heme catabolism is either used for biochemical purposes including translational regulation, or is stored or exported via ferroportin (FPN1) and delivered to transferrin. Iron is delivered to liver parenchymal cells by transferrin or by macrophage-derived L-ferritin . Lowering elevated intracellular heme via FLVCR-mediated export to HPX is another solution. However, this route would be compromised when circulating HPX is low or absent; and exacerbated when the hydrophobic drug binding site in HSA . The catalytic capacity of HOs may become overwhelmed by the amount of intracellular heme or when there are insufficient substrates and coenzymes for catalytic activity . Bone marrow transplantation successfully replenished normal progenitors of macrophages restoring Kupffer cell activity to the HO1−/− mouse liver  that entails extracorporeal blood circulation, including priming of the circuit with stored blood and potentially also blood transfusions. Patients with compromised liver function, especially liver cirrhosis and a history of alcohol abuse will not readily synthesize HPX . 19 molecules in 2.23 L av. plasma volume based on 55% of 4.7 L blood volumeNotably, plasma schistocyte count and urinary hemosiderin, a measure of excess RBC destruction, correlate inversely with HPX levels ; whereas Hp levels were related to Hb levels not heme . Sepsis can also entail prominent episodes of hemolysis that are ameliorated by HPX supplementation (see Section Key Roles for Heme Toxicity in the Pathogenesis of Sepsis and Protection by Hemopexin). Assessment of the association of HPX levels with the presence of specific diseases, response to therapies, and prognostication of clinical outcomes requires longitudinal studies. These involve repeated monitoring of risk factors and/or outcomes, often on a daily basis. Ideally, the controls will possess similar baseline characteristics as cases, with the primary differences being only the exposure of interest. Both HPX and Hp levels can change quickly in response to a hemolytic episode that “resolves” generally within 1–2 weeks. To a certain degree, the early research on HPX and Hp provides a unique window on the unfolding events affecting the metabolism of these proteins during and after hemolysis—and in the presence and absence of infection. The circulating plasma levels of Hp, HPX, and HSA vary quite widely within the normal population, but are stable for a healthy individual. Cohort studies that use data from much larger groups of patients, which share common characteristics and exposures, are the backbone of current proteomics and outcomes research. Large group cohort studies can be carried out prospectively or retrospectively with differing exposures to a suspected factor or treatment; and then observed and tested generally. Given the relatively rapid changes in HPX and Hp levels, frequent sampling may be necessary to identify correlations between changes in HPX and Hp metabolism and a patient's clinical status.Decreased plasma HPX levels in hereditary hemolytic diseases was first reported in a study of 38 children in 1963  within 24 h decreasing to normal after 6 days and HPX levels start to increase within 24–48 h. However, Hp remained low requiring 14 days to return to normal plasma levels. This was attributed to a slow ongoing destruction of RBCs and/or the absence of one or more sufficient stimuli for de novo Hp synthesis. As described in Section The Clinical Relevance of Hemoglobin, Hemopexin, and Haptoglobin Levels, macrophage clearance of RBCs is vital for heme and iron homeostasis as it minimizes oxidative stress and inflammation from heme toxicity  was seen in hemorrhagic pancreatitis, where plasma heme was high at 47 mg/100 ml Sears, . Acute hNormal or only slightly decreased HPX levels with low Hp were detected in paroxysmal nocturnal hemoglobinuria and in auto-immune hemolytic anemia  needed for normal flexibility of red blood cell membrane. These misshapen cells are recognized and taken up by the spleen and destroyed, often necessitating multiple blood transfusions. HPX levels were found to be normal (400 μg/ml) and Hp was low but rose ~5-fold to 600 μg/ml after splenectomy . Thus, to further define the stages and cause of hemolysis and particularly to distinguish impairment of heme clearance from hemolysis, knowledge of plasma heme and HPX levels would enhance the ability of clinicians to define the clinical ramifications.Overall, these and other clinical studies  demonstrate a need to examine the often stated concept, originating from the late 1960's, that HPX is depleted only when Hp is absent  in IVH from mechanical damage due to aortic and mitral prostheses; IVH from paroxysmal nocturnal hemoglobinemia due to complement destruction of RBCs; and HPX depletion (in the author) after five injections of hematin i.v. , with decreased neuromotor function . Cells rely on the proteins in extracellular fluids to limit such radical chemistry by converting pro-oxidant forms of iron to less reactive forms  sensitized porcine aortic endothelial cells in vitro to subsequent oxidative stress leading to cell death  were toxic. When heme was bound by HPX  there was no endothelial cell toxicity  are induced in mouse hepatoma cells within 3–6 h exposure to heme-HPX, whereas the iron released from heme catabolism may not induce ferritin via translation until ~7–11 h  and organ infarction. The only cure for SCD is bone marrow transplantation, which carries significant risks of morbidity and mortality due to immune suppression or injury induced by the donor immune system recognizing recipient antigens as foreign (graft vs. host disease). Other patient treatments include approaches to avoid vaso-occlusion crises, palliative therapy to manage pain and to relieve symptoms, and antibiotic prophylaxis to prevent infections and pneumonia. New treatments include gene therapy to produce normal Hb, drugs to produce fetal Hb , NO therapy to prevent sickle cell clumping and statins to limit cholesterol levels and decrease oxidative stress in blood vesselsThe extent of any hemolysis is driven by RBC instability and the extent of potential heme toxicity by changes in heme that affect it's binding to globin. Compared with HbAA mice, the RBCs in HbSS mice turn over rapidly (t1/2 of 2–5 days) in a steady state of stasis at ~8–10%. Infusions of Hb in the HbSS mice increased stasis to 30%. Co-administration of either Hp or HPX with Hb prevented stasis, supporting that heme is the active agent. HbS caused more stasis than HbA consistent with its known instability and supporting that HbS more readily releases heme; thus, enhancing the pathology of SCD. Infusions of HbA into control mice generated low levels of stasis . Thus, heme induces stasis in sickle, not WT, mice. As platelets and RBCs adhere to the walls of endothelial cells lining blood vessels, the blood flow slows and vaso-occlusion can occur. Heme must be released from metHbA to cause the pathology because the stable derivative of heme, cyanometHb, did not cause vaso-occlusion ; nor did mixtures of HbA with methylene blue (ferro-Hb), or HbA with Hp, or HbA with HPX.HPX deficiency in SCD mice promotes vaso-occlusion  formation, which was limited by HPX  and also infusions of immunoglobulins for certain immune deficiencies. Japanese and German physicians pioneered Hp infusions to prevent renal damage from hemoglobinuria in burns and, in 1977, the severe hemolytic damage accompanying ABO incompatibility  in survivors  heme toxicity can be central to the pathogenesis of a human disease allowing heme toxicity to develop as HPX levels decline ; and (ii) HPX infusions restore circulating HPX levels, reduced mortality, and thus may be a novel therapeutic approach came from studies using mice models of sepsis . HPX also protects cells from “heme sensitization,” the apoptotic cytotoxic effect of heme that requires exposure of cells not only to heme but subsequently to pro-inflammatory molecules such as TNF. Uptake of heme-HPX by primary human or mouse hepatocytes did not raise ROS levels as did heme, even after subsequent incubation with TNF for 16 h  and i.v. heme promotes tissues damage and leads to a severe “high grade” sepsis  and had further decreased but were still detectable in the Hmox1−/− mice Mice start to die as early as 2 days after CLP; however, HPX infusions (50 mg/kg ip) caused a significant decrease in this morbidity from 75 to 20% when given at 2, 12, 24, and 36 h post CLP. The control mice were given IgG (50 mg/kg ip) and an equal volume of the solvent control, PBS.Low-grade sepsis in response to colon puncture and ligation is toxic in Hmox1HPX together with other biomarkers such as Hp, may prove useful as a supplement in certain stages of hemorrhagic shock and even, potentially in the progression of Ebola in patients, especially those that progress to hemorrhage.Although hemorrhagic shock differs from septic shock  is a primate model with the most similar genetics, physiology, and metabolism to humans. In contrast, with respect to the response to infection, mice can tolerate far larger burdens of bacteria than humans without succumbing and although this differential response can be bacterial species specific, there are differences between humans and mice in their response to inflammatory conditions  and microvasculature (PMVECs), and dermal microvasculature were incubated with multiple exposures to heme (5–25 μM), considered not toxic, in fresh media every 48 h for 7 days. Five genes differentially regulated by heme in PMVECS and PAECs were shared: HO1, glutamate-cysteine-ligase modifier subunit, NQO1, H-ferritin and δ-aminolevulinate synthase 1. Nrf2 regulation was considered central: whereas HO1 was quickly induced by heme, NQO1 required several days and was apparent only in the older mice perhaps due to the oxidant burden of the disease and low levels of endothelial SOD. NQO1 staining was more intense in clinical SCD than in mice. One similarity between the mice and clinical SCD was the intensity of HO1 staining in post-mortem lung endothelium, although HO1 mRNA levels were highly variable in the patients. Plasma HO1 levels from SCD patients varied considerably, but correlated with markers of endothelial cell activation and injury. These enzyme changes may provide a strategy for additional SCD therapy  HPX sequesters heme from cells preventing direct cytotoxicity and heme sensitization; and in cells lacking HPX receptors this prevents endothelial and immune cell activation leading to stasis and exacerbation of the inflammatory response, respectively; (2) Plasma HPX targets heme principally to the liver and normally recycles intact after receptor-mediated endocytosis while the heme is degraded and its iron re-claimed; (3) Hepatic uptake of heme-HPX is via a high affinity low capacity system, likely LRP1/CD91, and a low affinity high capacity system  providing accurate diagnosis of acute illness; (2) improving our ability to determine which patients are at greater risk for more severe illness, complications, or death; and (3) developing targeted replenishment therapies for patients suffering from acute illnesses due to, or exacerbated by, significant hemolysis. However, large gaps remain in our understanding of heme metabolism and the role that HPX and Hp could play in specific disease states. In addition to the bench-side experiments using animal models that have provided much of the knowledge to date on the roles of HPX and Hp in heme and Hb metabolism, respectively, translational approaches that use human-derived samples from patients with and without specific diseases and/or risk factors are warranted.Future studies in human subjects should focus on several common diseases that often feature, or are complicated by, hemolysis. These diseases include SCD, sepsis, hemolytic anemias, and viral hemorrhagic fevers . Sepsis and SCD in particular have readily-available animal models and baseline data, and disease incidence is sufficiently common to allow both acute and longitudinal studies in humans. Future studies should be conducted to determine HPX levels in these disease states, to assess the discriminatory ability of HPX for disease in symptomatic and/or at-risk individuals, and to determine the prognostic utility of obtaining HPX levels in acutely-ill patients. When assessing severe illness characterized by prominent hemolysis, physicians currently use hematological profiles that quantitate cellular components and measure coagulation function. We propose that including plasma heme and HPX measurements as adjuncts to these standard hematological assays may further enhance the diagnostic and prognostic capabilities of the hematological profile, thereby enhancing clinical decision-making. An international standard for plasma heme and HPX, as previously suggested (Delanghe and Langlois, Both authors wrote and edited the manuscript; AS designed the figures.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.


Objective: To establish a safety profile for amnion-derived cellular cytokine solution following topical application in patients undergoing whole breast radiotherapy for breast cancer. Materials and Methods: Twenty female patients with early-stage breast cancer were enrolled in 2 separate cohorts of an institutional review board–approved phase I protocol. Cohort 1 consisted of 10 patients who received topical amnion-derived cellular cytokine solution to the breast immediately following the first 10 fractions of whole breast radiotherapy. Cohort 2 consisted of 10 additional patients who fit the same criteria as the initial cohort but received topical amnion-derived cellular cytokine solution following the development of at least grade I breast erythema. Blood samples were tested for the presence of proteins in amnion-derived cellular cytokine solution as well as routine hematologic functions. Results: Amnion-derived cellular cytokine solution did not induce overproduction of any cytokines sampled, and there was no evidence of “cytokine storm.” It also showed no significant absorption systemically following topical delivery. No patients developed an adverse event. There were no patterns of changes in vital signs or clinical laboratory tests that were related to the treatment regimen. Conclusion: In this safety trial, the topical application of amnion-derived cellular cytokine solution in both intact and denuded, irradiated skin was found to be safe, and showed no evidence of systemic absorption. No cosmetic changes were identified long term. Patient blood chemistry was not adversely affected, indicating the absence of an anaphylactic response and no evidence “cytokine storm” was identified. Amnion-derived cellular cytokine solution is safe to use topically in clinical protocols. The platelets release the contents of their α granules containing many growth factors that act as mitogens and chemoattractants. Platelet releasates and platelet-rich plasma have been used with some degree of success, but there is no homologous product currently available. Single growth factors made by recombinant DNA technology such as platelet-derived growth factor (PDGF), transforming growth factor beta, keratinocyte growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor (VEGF) have been used in clinical trials of wound healing, but only PDGF is approved for dermal wounds.1,2Amnion-derived cellular cytokine solution (ACCS), made by Stemnion, Inc , is a multiple cytokine solution derived only from a unique population of naturally occurring cells isolated from the full-term placenta that is normally discarded following delivery of a baby. Each placental donor has signed an informed consent and was screened for known infectious agents. There are no living cells in ACCS, only the multiple proteins, and there has been no manipulation of the genetic cellular material. ACCS has been found to contain multiple proteins involved in wound healing including VEGF, PDGF, angiogenin, transforming growth factor β, and tissue inhibitor of metalloprotease (TIMP)-1 and TIMP-2.12 was shown to be safe.3-5Independent collaborative studies have established proof of concept to enhance wound healing and tissue repair in both elective and traumatic wounds such as burns.-9There may be applications for ACCS in many different types of wounds. Improved healing in a natural physiologic manner has been demonstrated: for example, accelerated healing of surgical incisions, more rapid healing of second- and third-degree burns with decreased scarring, and reduction of the inhibition to normal healing caused by infection.4Twenty female patients with early-stage breast cancer between the ages of 18 and 80 years and who required postoperative radiotherapy were enrolled in 2 separate but related cohorts of an institutional review board–approved phase I protocol. Patients were excluded if they had abnormal hepatic or renal functions indices , were on hemodialysis, or were pregnant or lactating postpartum. Cohort 1 consisted of 10 patients who received topical ACCS to the breast immediately following the each of the first 10 fractions of whole breast radiotherapy. Cohort 2 consisted of 10 additional patients who fit the same criteria as the initial cohort but received topical ACCS following the development of at least grade I breast erythema as defined by the Common Terminology Criteria for Adverse EventsBefore entering the study, the subjects underwent lumpectomy for early-stage breast cancer. Whole breast irradiation was instituted between 4 and 12 weeks postlumpectomy. For patients receiving chemotherapy, whole breast irradiation began 2 to 4 weeks following the final cycle of chemotherapy. No patient received neoadjuvant chemotherapy. The whole breast irradiation dose was either 50 Gy at 2.0 Gy per fraction or 50.4 Gy at 1.8 Gy per fraction. Subjects were treated using standard immobilization techniques and 3-dimensional conformal, forward planned radiotherapy. A boost to the lumpectomy cavity plus margin of 10 to 16.6 Gy in 5 to 9 fractions was used at the discretion of the radiation oncologist.All safety data, including vital signs, were reported and selected data were by descriptive statistics. Summaries were prepared for the number (percentage) of patients with adverse events (AEs) and serious adverse events (SAEs). Quality of life was assessed globally as well as locally by noting changes in both the medial and lateral aspects of breast including breast texture (thickening and hardness), pain, tenderness, shape, sensitivity, swelling, redness, itching, flaking skin, blistering, and fluid leak.Planned statistical hypothesis testing comparing ACCS and saline across treatment regimens was not done because the small sample size would result in limited power to detect even moderately large treatment effects. We did record what were felt to be noted differences in the separate sides of each breast when differences did occur. These will be reviewed subsequently.The primary endpoint of this safety and efficacy trial was to determine the safety profile of ACCS as measured by AEs and SAEs. The secondary endpoints were quality of life, measuring the time until the onset of erythema (inflammation), reduction in erythema in comparison of each side, reduction in moist desquamation, and time to healing comparing skin treated with ACCS versus saline placebo control.Using this assay platform, ACCS did not activate peripheral blood mononuclear cells (PBMCs), affect their viability, or induce proliferation over a 72-hour culture period. Furthermore, the analysis of culture supernatants indicated ACCS did not induce overproduction of any cytokines associated with “cytokine storm”  measured at the same time-points  and 2. IAdverse events were nearly evenly distributed between ACCS-treated and saline-treated areas of the skin of the breast. There were no patterns of concern upon review of the AEs . Of the -14Acute radiation dermatitis is a frequent complication of whole breast radiotherapy. When the degree of dermatitis exceeds grade II as defined by the Common Terminology Criteria for Adverse Events,12,2,4-The use of ACCS has shown healing properties in some preclinical and clinical studies.1There are no comprehensive guidelines on the approaches required for immunogenicity testing during product development. Likewise, there is no single source of requirements for nonclinical or clinical studies and no specific guidance for interpreting assays. Therapeutic proteins are frequently immunogenic in animals; however, immunogenicity in animal models is not always predictive of immunogenicity in humans. Animal studies may not be helpful in determining the propensity of humans for hypersensitivity responses to protein therapeutics. However, assessment of immunogenicity in animals may be useful to interpret nonclinical toxicology and pharmacology data. In addition, immunogenicity in animal models may reveal potential antibody-related toxicities that could be monitored in clinical trials. It is essential to adopt an appropriate strategy for the development of adequate screening and confirmatory assays to measure an immune response against a therapeutic protein(s). Assays should be capable of distinguishing neutralizing from non-neutralizing antibodies, be validated, and standardized.,Consequences of antibody response to a biologic treatment modality fall into 3 categories: hypersensitivity reactions, reduction in efficacy (not considered in this phase I study), and induction of autoimmune diseases. First, hypersensitivity would involve IgE antibody production following administration of a protein therapeutic. Anaphylactic findings of hypersensitivity are easily identifiable by complete blood cell count blood work in a phase I study,16,21-In vitro methodology using human naive PBMC effector cells were used to determine immunogenic effects of ACCS. PBMCs act as sentinels to immunogenic substances, through activation, proliferation, and cytokine production. Through these monitors, the extent of immunogenicity of a biologic protein or protein solution can be determined.1925 This indicates ACCS did not elicit a sensitization reaction in guinea pigs. The fact that ACCS did not induce any skin irritation, even in primed test animals, indicates there was no activation of a primary or secondary immune response.ACCS was also examined for immunogenic potential using a relevant animal model that measures sensitization to biologic compounds. This method, known as the Buehler method, is a standardized model that uses guinea pigs to measure a substance's ability to induce both an immune response in the skin and a secondary immune response upon reexposure to the test material after a period of quiescence.In addition, whole-blood PBMCs were analyzed for activation markers CD25 and CD69 on the same samples to examine potential posttreatment activation of immune effector cells. These markers are usually expressed on activated populations of T lymphocytes and other WBCs. An increase in expression of these markers correlates with an elevation in cell activation. Immunogenic compounds, such as proteins, can induce expression of these markers on cells over time.Likewise, there were no significant differences between pre- and post-ACCS treatment PBMC expression in the activation markers CD25 and CD69. Together, these data are supportive in determining the safety of ACCS administered topically for treatment of inflammatory skin conditions. The fact that ACCS, when administered in this manner, is not absorbed systemically provides evidence supporting these data. Relevant findings of this safety trial including ACCS showed no significant absorption systemically following topical delivery.In this safety trial, the topical application of ACCS in both intact and denuded irradiated skin was found to be safe and showed no evidence of systemic absorption. There was a definite difference in the appearance of the sides of the breast in a number of women, but it did not correlate with application of ACCS following unbinding. Further investigation is warranted to evaluate the relationship of skin reaction to ACCS application due to this very stark delineation of effect. Patient blood/hematology/chemistry  were not adversely affected, indicating the absence of an anaphylactic response. No evidence of increase of the 9 inflammatory cytokines was seen and no evidence of “cytokine storm” was identified. ACCS is safe to use topically in clinical protocols. This study has demonstrated safety, and phase II and III clinical trials may proceed from this point.


Although cell adhesion is thought to fasten cells tightly, cells that adhere to each other can migrate directionally. This group behavior, called “collective cell migration,” is observed during normal development, wound healing, and cancer invasion. Loss-of-function of cell adhesion molecules in several model systems of collective cell migration results in delay or inhibition of migration of cell groups but does not lead to dissociation of the cell groups, suggesting that mechanisms of cells staying assembled as a single cell cluster, termed as “cell collectivity,” remain largely unknown. During the formation of Kupffer's vesicle , KV progenitors form a cluster and migrate together toward the vegetal pole. Importantly, in this model system of collective cell migration, knockdown of cell adhesion molecules or signal components leads to failure of cell collectivity. In this review, we summarize recent findings in cell collectivity regulation during collective migration of KV progenitor cells and describe our current understanding of how cell collectivity is regulated during collective cell migration. Individual cells have the potential to migrate randomly. When chemo-attractants are distributed in an environment, the cells can migrate toward the attractants. In some situations, cell aggregates can move directionally while maintaining cell adhesions in a process called collective cell migration. Collective cell migration is essential for the generation of basic organ structures such as sheets, clusters, spheres, sprouts, and vesicles in the morphogenetic processes of animal development Rorth, , and is Drosophila border cells  is a key organ required for the left-right asymmetric patterning in zebrafish  Figure  Essner . About 2cadherin1 and cadherin2  are expressed in DFCs , cadherin1 expression increases in migrating DFCs , 0 hpf, cas Figure , suggests Figure . On the s Figure . This mecadherin1 is knocked down specifically in DFCs, the DFC cluster is broken up into multiple groups of cells at 60–80% epiboly stages (6.5–8.5 hpf)  positive feedback regulator canopy1 in zebrafish and found that FGF-signal dependent regulation of cadherin1 expression is required for generating proper cell collectivity in the DFC cluster , one of the lateral giant larvae genes initially identified as tumor suppressor genes in Drosophila, is expressed in DFCs  bind to the intercellular domain of cadherin1 and interfere with the link between cadherin1 and β-catenin, leading to the inhibition of cell-cell adhesion  regulator, leads to failure to compact the DFC cluster during late-gastrulation stages (9–10 hpf)  Table , suggestA DFC-specific gene knockdown approach in which morpholino is injected into the yolk of embryos at the 256–512 cell stages has greatly contributed to our understanding of how DFC collectivity is regulated (this review), how DFC clustering occurs and whether DFC/KV morphogenesis is required for the establishment of left-right patterning  and finishes just 3 h , the EVL cells tightly bind to the yolk syncytial layer (YSL) and purse string contraction of the actin in YSL drives the migration of EVL toward the vegetal pole  breaks left-right symmetry. Focusing on cell collectivity regulation during KV organogenesis, we now understand the importance and the regulatory mechanism of cell collectivity in these processes. Furthermore, we show that cell adhesive properties change during collective DFC migration, indicating that multicellular tissues/organs are more dynamic than previously thought. Despite this substantial progress, many important questions remain. For instance, how do collective cell dynamics contribute to generating functional organs? How does the pairing of tight junction change? Are adherens junctions, tight junctions, and cell-ECM interaction coordinated? Does mechanical force contribute to collective DFC migration? Does collective DFC migration have analogy with other collective cell migrations seen in normal development, wound repair, and cancer invasion? It is of great interest to fill in these gaps to further clarify the regulatory mechanisms and importance of collective cell migration during organogenesis.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.


Hepatitis C virus (HCV) infection is one of the main causes of chronic liver disease. Viral persistence and pathogenesis rely mainly on the ability of HCV to deregulate specific host processes, including lipid metabolism and innate immunity. Recently, autophagy has emerged as a cellular pathway, playing a role in several aspects of HCV infection. This review summarizes current knowledge on the molecular mechanisms that link the HCV life cycle with autophagy machinery. In particular, we discuss the role of HCV/autophagy interaction in dysregulating inflammation and lipid homeostasis and its potential for translational applications in the treatment of HCV-infected patients. Hepatitis C virus (HCV) infection is a major global health problem. Almost 200 million people are infected worldwide, with 3/4 million new infections and 350,000 deaths per year , 2. HCV The HCV world is facing a revolution in terms of therapeutic approaches. First generation direct-acting antivirals (DDAs) are now available in clinics and a large series of new DDAs and host-targeted agents are in advanced clinical trials, with the expectation of curing more than 90% of infections . HoweverHCV does not cause a direct cytopathic effect on host cells; most of the related liver dysfunctions are considered a consequence of the virus's ability to dysregulate host processes . In partHCV is a blood-borne virus of the Flaviviridae family that replicates primarily in hepatocytes. The HCV genome is composed of a positive, single-stranded RNA of approximately 9.6 Kb, containing a long, open reading frame (ORF) flanked by nontranslated regions (NTRs) at 5′ and 3′ ends that regulate RNA translation and replication . HCV RNAHCV entry in hepatocytes is mediated by the interaction of the viral envelope proteins with several cell surface receptors and entry cofactors including glycosaminoglycans, LDL receptor, tetraspanin CD81, scavenger receptor class B member I, tight junction proteins claudin1 and occludin, and the cholesterol uptake receptor Niemann-Pick C1-like 1 . HCV parHCV RNA is delivered to endoplasmic reticulum, where viral translation is mediated by an internal ribosomal entry site located in the 5′ NTR. Among nonstructural proteins, NS4B and NS5A are able to induce ER membrane alteration, called membranous web, that provide a scaffold for the assembly of the HCV replication complex and protection from host immune defenses , 19. HCVThe term autophagy refers to a series of processes that allow for the degradation of intracellular components by delivering them into lysosomes . In mostAlthough autophagy was originally described as a nonspecific process induced in response to nutrient deprivation, it is now evident that many selective forms have evolved to recognize and degrade damaged, supernumerary, or unwanted substrates. Consistently, signaling pathways triggering selective autophagy as well as substrate receptors specific for unique targets have been identified . For exaThe emerging complexity in the field of selective autophagy provides new hints for understanding how autophagy alterations could be associated to the development of a wide range of human diseases –43. In tHCV is highly successful in establishing chronic infection. Since 2008, several studies have been conducted to clarify the contribution of autophagy alterations in establishing chronic HCV infection and in the onset of associated diseases –49. In pThe emerging scenario, not devoid of conflicting results, indicates the presence of a delicate balance between proviral and prohost functions of autophagy depending on the activation of selective types of this process. in vitro infection or replication systems, proper fusion between autophagosomes and lysosomes has been reported, as well as the occurrence of LC3 protein degradation within the lysosome, indicating that a functional autophagy flux occurs in the presence of HCV [The presence of an increased number of autophagosomes in HCV-infected cells has been reported by several groups, based on observations in either cell culture infection systems or liver samples from patients with chronic HCV infection –55. Howee of HCV . A possie of HCV , 57  stress . HCV infThe activation of signaling pathways downstream to ER stress may play a role in the induction of autophagy by HCV. For example, it has been proposed that the induction of autophagy by HCV depends on the inhibition of the AKT/mTOR pathway by UPR . HoweverAn important consequence of ER stress induced by HCV is the alteration of ER calcium homeostasis . ReleaseA central role of mitochondria in the induction of autophagy has been also confirmed by a recent study aimed at characterizing common mechanisms of autophagy modulation by different RNA viruses , 78. TheIt is therefore likely that a combination of different pathways, not all yet characterized, may induce autophagy at different stages of HCV infection. HCV infection activates different pattern recognition receptors , which aIn this regard, an integrated analysis of how autophagy is modulated during different steps of infection will provide useful information to elucidate how HCV intersects the autophagy pathway.Several reports have highlighted a strict interdependence between the execution of the HCV life cycle and autophagy activity or the expression of autophagy proteins. It is recognized that inhibition of the autophagy machinery affects the production of new infective particles , 69. HowHCV replication was shown to occur either normally or drastically inhibited in autophagy-deficient cells , 59, 69.The highly heterogeneous nature of Huh7 cells and derivative clones and the different strategies of HCV expression (RNA transfection versus infection) may account, at least in part for the observed discrepancies. In addition, the interaction between HCV and the autophagy machinery may be regulated in a temporally regulated manner, as suggested by a recent study showing that the colocalization of ATG5 and NS5B is detected at 2 days postinfection, while it is absent at 5 days . This hyHCV has evolved multiple mechanisms to evade innate immunity. In particular, HCV inhibits the interferon (IFN) response, acting both upstream and downstream to IFN production . For exaβ production by autophagy was dependent exclusively on the interaction between the Atg5-Atg12 complex and the RIG-I and IFN-β promoter stimulator 1 (IPS-1) proteins [A turning point in elucidating the relationship between autophagy and HCV infection is represented by three recent works demonstrating the involvement of autophagy in repressing the anti-HCV innate immune response . Indeed,proteins .Although the relationship between autophagy and innate immunity in HCV infected cells has been well established, it remains unclear whether the exacerbation of innate immune response is the main cause of the inhibition of HCV infection in autophagy-defective cells. To answer this question, the impact of autophagy inhibition on HCV infection should be tested in cells defective for the major viral sensing pathways.α) receptors but not type II (IFN-γ) or type III (IFN-λ) [ in vivo [ER stress and autophagy are also involved in the inhibition of the antiviral response downstream to IFN production by downregulating the expression of type I IFN (IFN- (IFN-λ) . In the  in vivo . On the  in vivo , 98, whiβ is able to trigger autophagy-mediated degradation of the viral protein [β, since it is not observed when mice are treated with IFN-α. If confirmed in the context of HCV infection, these results represent an important example of how autophagy may play both positive and negative roles in the innate immune response to HCV infection. In relation to these results, it has been reported that HCV clearance can be induced by treating infected cells with AICAR, an AMP analog capable of activating AMPK, a protein kinase with pleiotropic functions including direct stimulation of the autophagy process [Recent data highlighted a further level of complexity in the crosstalk between the interferon pathway and autophagy in the context of HCV infection. By means of transgenic mice expressing HCV NS3/NS4A protein in the liver, it was shown that IFN- protein  (Figure  process . HoweverHCV infection is not only impaired by the general inhibition of the autophagy process but also by interfering with the induction of mitophagy, a selective type of autophagy activated by the PINK1/Parkin pathway , 77.In light of the autophagy-innate immunity relation, it would be important to test whether mitophagy is also able to hamper the innate immune response. Interestingly, aberrant stimulation of mitochondrial fission by HCV, which leads to mitophagy, results in the repression of interferon response . AlternaThe HCV life cycle is tightly coupled to the lipid metabolism of host cells , 19. HCVHCV not only associates with intracellular lipid compartments but also induces profound alteration in lipid metabolism leading to steatosis . The cor in vitro studies using both HCV-replicon and HCV-infected cells. In these systems, we found that a large fraction of autophagosomes selectively colocalize with lipid deposits, whose levels are increased by viral replication [Lipophagy has been recently described as a selective type of autophagy dedicated to the degradation of intracellular lipid stores , 112. Thlication  (Figure lication . Taken tMany aspects of the role of lipid-selective autophagy in HCV-infected cells require further investigation. For example, it will be interesting to test whether the interaction of core protein with lipid droplets, in particular from HCV genotype 3 with a higher steatogenic potential, is able to interfere with the access of these structures to the autophagic machinery. Moreover, being cholesterol essential for HCV virion assembly and release , it is cThe dual role played by autophagy in inflammation and lipid metabolism in HCV-infected liver cells raises an important question regarding the contribution of autophagy defects in disease progression towards steatohepatitis, fibrosis, cirrhosis, and hepatocarcinoma in patients with chronic HCV infection. Recent studies not focused on HCV infection show that the onset of steatohepatitis in nonalcoholic fatty liver patients is associated to lower levels of “bulk” autophagy and, in particular, to the accumulation of the autophagy cargo protein p62 –115. In Autophagy has been proposed as a potential target for tackling HCV infection. However, the emerging scenario indicates that many types of selective autophagies are playing a role in the liver homeostasis of both healthy and HCV-infected individuals. Full elucidation of the molecular basis of this selectivity is therefore needed to provide a framework for the rational design of feasible anti-HCV drugs or intervening applications.


Background and Aims. The largely Mississippian strata of the Kilpatrick Hills, located at the western end of the Scottish Midland Valley, enclose several macrofossil floras that together contain ca 21 organ-species of permineralised plants and ca 44 organ-species of compressed plants, here estimated to represent 25 whole-plant species . The most significant locality is the internationally important volcanigenic sequence that is reputedly intercalated within the Clyde Plateau Lava Formation at Loch Humphrey Burn, where ca 30 m of reworked tuffs and other clastic sediments enclose one of the world’s most important terrestrial lagerstätten of this period. We here explore the palaeoecology and palaeoenvironments of the locality, and elucidate its controversial age.Methods. Repeated re-excavation of key exposures allowed recognition of five main depositional units, differing in thickness from 4 m to 12 m. It also permitted detailed sampling for plant macrofossils and microfossils throughout the succession. Several approaches are integrated to re-assess the taphonomy and preservation of these exceptional plant fossils.Key Results. The deposits are rich in taxonomically diverse miospores and in toto contain at least six well-developed compression floras, together with two beds yielding nodules that enclose well-researched anatomically preserved plants permineralised in calcite. Bulk geochemistry shows that the upper nodules formed by migration of Ca with subordinate Mn and Na. Some phylogenetically important plant fossils recovered in the early 20th century have been traced to their source horizons. Trends in relative proportions of macrofossil and microfossil taxa through the sequence are only moderately congruent, perhaps reflecting the likelihood that microfossils sample the regional rather than the local flora.Conclusions. The Loch Humphrey Burn sequence encompasses a wide range of depositional environments that intercalates high-energy fluvial channels  with lower energy flood plains and a brief lacustrine interval; all yield macrofloras typically dominated by allochthonous pteridosperms. The uppermost unit represents clastic swamps dominated by (hypo)autochthonous lycopsids and ferns s.l., and is tentatively correlated with the entire—reputedly mid-Visean—exposure at nearby Glenarbuck. Other nearby localities with rooted tree-lycopsids appear to have immediately pre-dated the onset of regional volcanism. These interpretations allow revised provenancing and dating of historical collections of key plant fossils. The late Tournaisian date previously attributed on palynological evidence to the lowest unit at Loch Humphrey Burn appears increasingly improbable when our re-appraisal of the macrofloras and microfloras is placed in the context of (a) statistical comparison with other permineralised Mississippian assemblages and (b) recent stratigraphic and geochronologic studies in the region; rather, we ascribe the entire Kilpatrick Hills sequence to the mid-Visean. Stratigraphic and palaeoenvironmental interpretations of the Mississippian rocks of the Kilpatrick Hills have especially profound implications for our understanding of the physical evolution of Scotland during the Variscan orogeny and formation of Pangea. The Geological Conservation Review for Palaeozoic palaeobotany ; they arThese three premium fossiliferous localities, which can legitimately be described as exceptionally preserved ‘lagerstätten’, together span much of the late Tournaisian and Visean . They weThis paper presents a detailed account of the plant-bearing localities of the Kilpatrick Hills, focusing on stratigraphical and palaeobotanical information gained from an intensive excavation in 1985 by RMB of the Loch Humphrey Burn locality. This was described by Relative dates derived from plant macrofossil  and espeThus, the Loch Humphrey Burn site has important implications for our understanding of the reconstruction of Mississippian plants and of their phylogenetic relationships, the palaeoecology and palaeoenvironments of volcanigenic terrains, and the dynamic stratigraphy of Scotland through a critical period in its geological history, namely the Variscan orogeny.The rocks of the Kilpatrick Hills have long been recognised as being dominantly Visean in age  interpreted the reworked tuffs and other dominantly fluvial deposits that characterise the Glenarbuck and Loch Humphrey Burn sequences as evidence that they were transitional between the Clyde Sandstone sediments and the fully expressed volcanic activity of the Clyde Plateau Volcanic Formation. He invoked considerable vertical displacement of the small, fault-bounded, horst-like blocks evident in the local geology  to explac Member . ClarifyStigmaria was collected at the locality in 1872, and the presence of this organ-genus was soon reported in a general description of the area by Plant-bearing localities were first recorded in the complex volcanigenic sequences of the Kilpatrick Hills in about 1870. Field surveyors operating on behalf of the British Geological Survey first located the plant-bearing locality east of a waterfall in Glenarbuck Burn [NS452748] . A permiThe classic site for both permineralised and compression floras immediately south of Loch Humphrey Burn [NS468754], 1.3 km ENE of Glenarbuck, was probably discovered by the British Geological Survey at about the same time as Glenarbuck, although the earliest documentary record is a letter that accompanied specimens from Loch Humphrey Burn sent to Robert Kidston by James Bennie in 1886 . Much ofEarly field-workers exploring the Kilpatrick Hills e.g.,  briefly ca +300 m asl) and shales intercalated with thin, often discontinuous, organic-rich dark grey bands described in previous literature as ‘coals’ e.g.,  but bettdeposits .ca +300 m asl) ‘hanging valley’ into a 30 m-high fault scarp of tuffaceous sediments, creating two exposures (ca 60°) east-facing bank of the valley , a sphenopsid  and a single fragment of a fern phyllophore (Etapteris tubicaulis)   and 1E. Protocalamites, several fern taxa  means that their age is uncertain  account of the local palaeoecology, During April–May 1985, RMB crudely surveyed all the areas of volcanic ash in the Kilpatrick Hills indicated on the 6” field slips used by in situ and marking palaeosols . However, the more interesting exposures occurred along the westernmost lava scarp, Lang Craigs , where buff-coloured siltstones and fine sands with thin impersistent, organic-rich shales  crop out immediately below the lavas . These sediments include several horizons of calcareous concretions that vary considerably in size and degree of induration. Examination of numerous concretions revealed ubiquitous permineralised stigmarian rootlets, two weakly permineralised Stigmaria cf. ficoides root branches and a single fragment of a probable Lepidocarpon strobilus. These assemblages most likely represent contemporary wetland soils.Both of the ‘horst-block inliers’ indicated in However, plant assemblages from these localities were judged insufficiently diverse and/or well preserved to merit detailed study. Although the classic localities at Loch Humphrey Burn and, especially, Glenarbuck had become severely weathered and largely obscured by talus, RMB decided to concentrate a substantial part of his doctoral research  on Loch (1)Protopitys scotica, Calathospermum scoticum, Germinitheca scotica).Its diverse recorded flora of both compressions and permineralisations  includes(2)At least one plant-rich horizon demonstrably contained compressions and permineralisations preserved in intimate association, apparently presenting an exceptional opportunity to prove attachment of compressed and permineralised organs and thus at least partially reconstruct conceptual whole-plants cf. .(3)Alcicornopteris hallei–A. convoluta As previous palaeobotanical studies were based on a relatively small amount of material collected before 1935, it seemed probable that further collecting, preceded by re-excavation and followed by the application of modern analytical techniques, would not only provide new information on species known to occur at the locality but would also reveal species new to the Kilpatrick Hills (or even new to science).(5)A miospore profile spanning 30 m of sediment  had recePreliminary inspection of the classic Loch Humphrey Burn locality (Exposure A1) showed that a much more ambitious restoration programme was needed to elucidate its stratigraphy, identify the horizons collected by previous workers and enable collection of new material. This work was funded by the then UK Nature Conservancy Council; it was performed in September–October 1985 by RMB and two paid assistants, Robert Gray and Gordon Barrass: (1)A face 26 m long and up to 18 m high (Exposure A1) was manually cleared of talus and photographed  and 5.(2)Selected profiles cut back to unweathered rock provided three lithological logs Promising plant-bearing horizons identified during (2) were further investigated, and preliminary collections were taken from nine levels  that was logged to assess the degree of lateral variation in the various stratigraphic units. Profiles 2–4 can be compared with the lithological log for Exposure B published by Three profiles were recorded from Exposure A1  and 6. PThe detailed lithological logs presented in )Unit 1 contains a series of poorly laminated, poorly sorted, coarse, greenish-grey sandstones, rich in volcanic ash and often containing large (≤20 cm) rounded lava clasts; these are interrupted by only one thin shale.et seq.) with his three new species of woody stem; the poor quality of preservation of his Calamopitys radiata in particular strongly resembles that of stems extracted from the bed in 1985 by RMB This relatively thin unit consists of largely unfossiliferous, putatively late Tournaisian laminated siltstones and fine sands overlain by the putatively early Visean secondary limestone and dark grey argillaceous ‘Fish Bed’ of The calcareous nodules found in ‘Bed 6’ at Exposure B and in the equivalent bed at the base of Exposure A1 (Flora 2) contained small permineralised plant fragments. These were poorly preserved and unidentifiable at Exposure A1 but at Exposure B they yielded a diverse fern–sphenopsid–pteridosperm flora .These finer, prominently laminated sediments appear to have been deposited in a lower energy flow regime than the underlying conglomerates, and are poorer in recognisable volcanigenic clasts. The lower siltstones are probably dominantly alluvial flood-plain deposits, though the overlying secondary limestone and dark shale rich in animal fossils were interpreted as lacustrine by  mid-Visean; 3.65 m; ‘Bed 12’)ca 30 cm), flattened, brick-like structures consisting of thinly laminated silty shales are surrounded by several concentric layers of similar sediment but lack appreciable cementation. A similar but much thinner horizon occurs near the top of the unit . It is dominated by many of the species of pteridosperm foliage and reproductive organs that characterise Unit 4, though RMB also exhumed a single specimen of the arborescent lycopsid rootstock Unit 3 resembles Unit 2 in texture and lack of volcanic clasts, and is possibly also an alluvial flood-plain deposit. However, it has a much greater organic content that includes large, delicate plant fragments, suggesting that this was fed by a richer, and hence presumably more proximal, source of plant material.Unit 4 exhibits considerable lateral and vertical heterogeneity  and 6, tProtocalamites, some of which were partially permineralised and reached 2 cm in diameter.Unit 4 is the most palaeobotanically significant portion of the Kilpatrick Hills sequence; most of the constituent beds of this unit contain plant remains. Several beds, particularly in the upper part of the unit, yielded only fusainised fragments, but five contained readily identifiable material  they consist of 3 cm of brownish-grey shale, coarsening up into 7 cm of fine–medium sand  that is The shale at the base of Flora 4 could be traced reliably across the exposure  to the northern extremity, but the distinctness and richness of the associated uncemented fine sands decreased rapidly northward. This lateral change in lithology was accompanied by decreased quality and increased weathering of the compressions; both are probable consequences of the petering out of the overlying concretionary horizons  and 7.Flora 5 consisted of sporadic dark brown to black compressed plant fragments, mostly fern-like foliage, dispersed among several centimetres of green, medium-grained sandstones 1–1.5 m above Flora 4. These were cemented in Profile 4 but uncemented further north.ca 2.3 m above Flora 4, in 2–3 cm of brownish-grey indurated shale located immediately below several centimetres of greyish-green medium sand. This horizon could be traced for ca 16 m from the south end of the exposure , possibly the compressed equivalent of the permineralised L. kilpatrickensis described by The lowermost bed of Unit 5, a cemented greenish-grey medium sandstone, contains Flora 9 . This yiin situ stigmarian rootlets and other lycopsid remains. Poorly-developed palaeosols containing in situ rootlets occur throughout Unit 5, delimiting several periods of landscape stability and indicating the presence of persistent in situ lycopsid-dominated communities, probably occupying low-lying swamps that were subject to periodic inundation.Taken together, the overall facies of Unit 5 suggest a return from the channel-reworked volcanigenic sandstones of Unit 4 to lower energy alluvial flood-plain environments. However, this transition was evidently gradual; the lowermost 4 m of Unit 5 includes four medium–coarse sandstones that resemble in lithology those of Unit 4 but enclose The large number of blocks extracted from Unit 4 at Loch Humphrey Burn were slowly dried in the laboratory and then brushed free of surficial dirt. Subsequent cleaving along bedding planes attempted a difficult balance between the desire to expose as much plant material as possible  and the fear of causing excessive fragmentation along the two inter-penetrating sets of closely-spaced joints that characterised those blocks. Further exposure of compression fossils was achieved by dégagement using mounted needles. Cleaving occasionally revealed small concretionary nodules . Initially, the nodules from exposure A1 were removed from the matrix with care, but it soon became apparent that permineralised plants within the nodules could not be correlated with compressions on the surrounding bedding plane due to diagenetic disruption of the surrounding matrix during formation of the nodule. The bulk of the collection remains uncleaved. Supposedly Tournaisian blocks taken from Unit 1 in exposure A2 were less well-bedded and more indurated than the overlying Visean material. Numerous wood fragments (some large) were excised from the matrix, which was then discarded .c axis) and width (b axis) were recorded in mm. One end was arbitrarily designated ‘Top’, the opposite end ‘Bottom’. Nodules had often nucleated around stems or branches, so an initial cut perpendicular to the c axis of the nodule was most likely to provide a transverse section of the target plant organ(s). Subsequent cuts with thin-bladed saws (width of cut = 1.0–1.2 mm) usually paralleled the first cut and were spaced 8–10 mm apart. Each cut surface was subjected to peeling using 50 µm-thick acetate , has not migrated and thus should be a constant proportion of both the nodules and the surrounding less indurated sediment. The observed ratio of matrix SiO2 to nodule SiO2 (1.36:1) was then used to calculate the expected percentages of all other major element oxides and eight selected minor elements in the diagenetic nodules , and the percentage divergence in the nodule from the expected value was noted  and in what relative proportions. The approach used was developed by as noted .2 had not migrated, but in fact no negative deviations exceed 20% for the present data-set. Most of the positive deviations can similarly be discounted as falling within the presumed margin of error of this rough-and-ready method, but four oxides show significantly greater concentration in the nodules. Four-fold increases in MnO and Na2O, and a more modest increase in MgO, are evident, though all three elements occur at low levels relative to SiO2, Al2O3 and CaO. Most notably, CaO shows a 16-fold increase in the nodules relative to its low levels in the surrounding matrix; this presumed ionic migration is sufficient to account for the carbonate content of 28% in the nodules, relative to less than 1% in the surrounding reworked tuffs. The greatly increased CO2 content of the nodules suggests that much of the dissolved, migrating Ca crystallised out as CaCO3—an interpretation that was supported by the subsequent examination of petrological thin-sections, and explains the presence in the nodules of plant fossils showing good-quality calcareous permineralisation.Any strongly negative divergences would indicate violation of the key assumption that SiOHowever, despite the dominantly calcitic permineralisation, extensive acetate peeling of plant-containing nodules demonstrated that the anatomical detail that can be observed in acetate peels following etching in 5% HCl alone is more limited than that evident in petrological thin-sections . We suspParalycopodites, Lepidophloios), one sphenopsid , three ferns , and two probable lyginopterid pteridosperms , together with the much-discussed, but still taxonomically ambiguous, putative heterosporous lycopsid Mittagia.Glenarbuck has yielded a flora of at least nine whole-plant species that is relatively egalitarian at higher taxonomic levels: the organ-species contain the components of at least two rhizomorphic lycopsids  appears convincing. These records of rhizomorphic lycopsids from putatively mid-Visean Glenarbuck precede the better-established late Visean occurrences of these genera at Pettycur  studied the first-found flora, which we believe originated from the lower part of Unit 1 (the supposed late Tournaisian sequence) at Exposure A2 (Flora 1 of this study). Of the three stem-species described by DH Scott from this locality, one  closely resembles our own collections from Flora 1. A second stem-species, Bilignea  obtained diverse, internationally important compressions and nodular permineralisations from the mid-Visean Unit 4 at Exposure A1 (Flora 4), and very similar material was collected from a putatively corresponding stratigraphic level at Exposure B by Palaeobotanically, this study has concentrated on Floras 1 and 4. Flora 1, unknown since the end of the 19th century until rediscovered by RMB and R Gray in 1985, was extensively collected. Unfortunately, all of the numerous wood specimens that were found proved to be poorly preserved. Flora 3 was first discovered and collected at Exposure B by AC Scott, and additional material of this flora was collected from the classic horizon at Exposure A1 by RMB and G Barrass. Although better preserved than Flora 1, Flora 3 is prone to fragmentation, due to the close-spaced jointing of the sediments; also, its permineralisations are both very localised and relatively inaccessible. Nonetheless, Walton’s Flora 4 remains the jewel in the crown of the remarkable fossil floras of the Kilpatrick Hills.Protocalamites goeppertii—is shared between Units 2 and 4 , there is no doubt that the floras are radically different in taxonomic composition, even at class level; current evidence suggests that only one whole-plant species—that most commonly represented by the stem-species  2 and 4 .Calamopitys, though the unprovenanced putative lyginopterid pterodsperm stems Eristophyton and Bilignea are also tentatively attributed to this flora.The only plant definitely attributed to the permineralised wood assemblage of Unit 1 is the calamopitid pteridosperm stem Protocalamites. Also found have been the lycopsid megaspore-genera Lagenicula and Setosisporites  representing three orders sensuCladoxylon) with others that are more typical of the Visean and Pennsylvanian , and shows especially strong genus-level similarity with the late Visean flora at Pettycur. Not surprisingly, these two floras have figured especially prominently in discussions of the early diversification of the phylogenetically heterogeneous “ferns”  whole-plant species. Its composition, in terms of higher taxa, is more typical of permineralised Mississippian assemblages of the Midland Valley, being dominated—in terms of both species diversity and biomass—by pteridosperms cf. . It has hylogeny .Protopitys, but this species has been critical in circumscribing the order Protopityales  are particularly morphologically diverse, and have strongly influenced thinking on homologies with the female organs of more derived gymnosperms, as well as prompting the concept of a strongly multi-ovulate “megacupule”  is also instructive when attempting to estimate the whole-plant diversity of the flora  and 1D. Archaeocalamites and anatomically-preserved Protocalamites has become widely recognised  foliage. In contrast, the limited palaeobotanical material extracted from Unit 5 indicates domination by arborescent lycopsids, and thus more closely resembles the Glenarbuck flora .sensuReviewing the macrofossil data previously summarised by Bateman , Our next task was to determine whether the microfloras of the Kilpatrick Hills yielded floristic data congruent with those already obtained from the macrofloras.The palynology of fossil plant-bearing localities of the Kilpatrick Hills was first investigated by G Clayton in , who colper se , Punctatisporites (≤21%) and Stenozonotriletes (≤6%). The first two of these three miospore-genera were tentatively attributed to pteridosperm pollen-organs described from Oxroad Bay by Convolutispora species have also been found in filicopsids, and Punctatisporites in members of several major taxonomic groups. The sample from the Walton Bed in the centre of Unit 4 yielded the largest number of miospore genera (30), whereas the lower of the two samples analysed from within Unit 5 yielded the smallest number (10), albeit handicapped by the small number of miospores recovered.In total, the 11 palynological samples quantitatively analysed yielded a remarkable 53 genera of miospores : of thessensuca 3 m above palaeosols demarcated by lycopsid rootlets , Radiizonates , Raistrickia (sample 2), Camptotriletes and Retispora (both sample 8). In contrast, the three samples scoring highest on Axis 2 contain significant proportions (>5%) of otherwise rare filicopsid spore-genera, either Apiculatasporites (samples 6 and 7) or Grumosisporites (sample 11). Axis 3 (not shown) merely separates sample 7 from the remainder, largely on the basis of the presence of small quantities (ca 2%) of the lycopsid spore-genus Anapiculatisporites. Only occasionally do stratigraphically adjacent samples plot close to each other in The microfloral results show considerably less variation in genus-level composition across the sequence than do the macrofossils . This obca 3 m).The relative consistency of the miospore data suggests that the gymnosperm-only macroflora of Unit 1 (exclusively woody stems and branches) is highly unrepresentative of the regional vegetation, and is almost certainly a taphonomic ‘artefact’ of sorting in these coarse, high-energy sediments. Foliar and reproductive organs of pteridosperms are the most frequent macrofossils in the other high-energy, volcaniclastic-rich portion of the succession, Unit 4, but here again, this trend is not reflected in the microfossil assemblages. Also, sufficient contrast is evident between the macrofossil dataset gathered from the Walton Bed in the middle of Unit 4 relative to the dataset generalised across the whole of Unit 4 to suggest that finer-scale sampling of the macrofloras would have been advantageous. Both microfossil and macrofossil datasets agree that the upper part of Unit 5 is comparatively rich in ferns, but the increase in lycopsids suggested by the macrofossil data is not mirrored in the microfossils or earlier ;Unit 1, which contains an important flora (see ‘Unit 1: Green-grey ashy conglomerates’), must be CM palynozone (2)Scott, Galtier & Clayton  could be CM palynozone (Ivorian: late Tournaisian) or Pu palynozone (Chadian–Arundian: early Visean);The 1.5 m of sediment between the top of ‘Bed 6’ (which contains another important flora) and the base of ‘Bed 11’ (the ‘Fish Bed’) of (3)ca 3.65 m of dark grey shales that comprise Unit 3 could be Pu palynozone (Chadian–Arundian: early Visean) or TS palynozone (Holkerian: mid-Visean);The (4)de facto the locus classicus for this palynozone.The plant-rich Unit 4 is confidently attributed to the TS palynozone (Holkerian: mid-Visean), presumably along with the lycopsid-dominated palaeosols of Unit 5. The Kilpatrick Hills are AC Scott and colleagues later expressed considerable confidence than their earlier and more tentative conclusions: “This new research, which included more precise dating of the differing plant bearing levels, indicated that there were at least two floras of different ages (a new[ly found] late Tournaisian flora and a mid-Viséan flora). Subsequent palynological work indicated that the age of the Upper flora could be revised yet further to an early Upper Viséan age . The hisca 8 my of deposition between the late Tournaisian sediments below and the mid-Visean sediments above. Although this lacustrine bed presumably represents a more quiescent period of deposition compared with the rapidly fluctuating fluvial sedimentation above and below, it seems extremely unlikely that either (a) it would take ca 8 my for 20 cm of sediment to accumulate, or (b) such a long period of non-deposition could occur in a tectonically active terrestrial environment without detectable erosion taking place. It is more probable that the palynozones, most of which were originally established for Mississippian sediments in eastern Scotland, require revision and refining when they are applied to sediments located further west.” Indeed, based on these miospore dates, the entire section at Loch Humphrey Burn is inferred to represent ca 14 Myr  noted that “if Pu miospore assemblages do prove to be confined to the ‘Fish Bed’, this interval is inferred to represent a 14 Myr .Unsurprisingly, the miospore dates first published by sensuca 20 Myr period that happens to coincide with the entire spectrum of potential pollen dates attributed to the combined Loch Humphrey Burn and Glenarbuck sequence . Bateman  concluded that “these exposures are therefore identical with, and probably correlate with, the lycopsid-dominated uppermost beds (Unit 5) at Loch Humphrey Burn. This suggests that either (a) Units 1–4 are very localised, or (b) they are widespread but rarely exposed.”In addition to Glenarbuck and Loch Humphrey Burn, several other exposures of sub-laval “tuffs and agglomerates” in the Kilpatrick Hills examined during the present study revealed laminated greenish-brown siltstones and fine sands exhibiting several thin impersistent black organic shales and poorly developed palaeosols delimited by Bateman concluded that “geomorphological evidence supports the latter hypothesis.” Specifically, he noted that at most other palaeobotanical sites in the heavily glacially eroded Kilpatrick Hills, including Loch Humphrey Burn and Glenarbuck , plant-bAlthough studies of stratigraphy in the Hills based on borehole data and lava lithology permits the correlation suggested by Bateman  between Unit 5 at Loch Humphrey Burn and the entire Glenarbuck sequence, the sediments enclosing both sequences appear to be intercalated within the series of basaltic lavas that cap the Hills. In contrast, the majority of the other exposures, such as Lang Craigs , appear or younger” . These communities were dominated by lycopsid trees with an interstitial flora that included horsetails, plus a few specialist ferns and pteridosperms. In contrast, Units 1–4 at Loch Humphrey Burn record a rapidly changing series of communities dominated in terms of biomass by pteridosperms and progymnosperms, but with at least sporadic understorey vegetation that contained seemingly rotating suites of several fern species but retained the persistent and ecologically adaptable horsetails throughout. Occasional wildfires are evident, especially in Units 1 and 2. These fires could have benefited from the gradual elevation of atmospheric oxygen levels during the late Palaeozoic, though current estimates suggest that oxygen levels in the mid-Visean were only slightly higher than those of today, rising to a peak of ca 32 ± 2% only in the Permian  are those containing the smallest recorded numbers of organ-species , and areWith regard to dating the three Kilpatrick Units, it could be argued that the two Units widely regarded as being Visean in age are more similar to each other than either is to the lowermost, supposedly late Tournaisian, Unit 2. However, it is the central Unit 4 that occurs within a cluster of five indisputably late Tournaisian floras, even though stratigraphically it occurs between Units 2 and 5, which are closely associated with both Tournaisian and Visean floras . We suggThis interdisciplinary study has addressed a wide range of scientific questions, generating strong answers to some and tentative answers to others, but also raising yet more questions. Areas demanding further research are thus both numerous and diverse: (1)Thorough study of the large volumes of compression fossils extracted from the Loch Humphrey Burn locality could not only clarify the circumscription and taxonomic affinities of the organ-species present but also lead to a better understanding of the palaeoecological transitions that are undoubtedly captured in this sequence.(2)Although the permineralised floras from Loch Humphrey Burn have been more thoroughly studied than the co-occurring compressions, there remains considerable potential for strengthening the existing tentative correlations of key organ-species across preservational states cf. .(3)Bilignea plant—clearly an ecological dominant of the period—lies in combining data from Loch Humphrey Burn (present study), East Kirkton The continuing uncertainty regarding the stratigraphic origin of two of the organ-species of pteridosperm stems studied by (5)Sampling of miospore assemblages from various lycopsid-dominated outcrops immediately below the lowermost Clyde Plateau lavas would be useful, to challenge the supposed CM date of the overlying volcanigenic sediments, and particularly to test our prediction that the subvolcanic sediments should straddle the Pu–TS palynozone boundary .(6)Our interpretation of the complex stratigraphy of the Kilpatrick Hills has depended on radiometric dates from structural blocks to the southwest; it is highly desirable that such dates are also obtained in the Kilpatrick Hills Block—specifically, from lavas both below and above the Loch Humphrey Burn sequence. The volcanic stratigraphy of the area might also be further refined through comparison of the detailed geochemistry of successive lava flows.(7)Given the substantial number of palaeobotanical localities already identified in the Kilpatrick Hills, we suspect that a more intensive field survey of the region would reveal additional, potentially valuable, plant-bearing outcrops.In conclusion, further work toward reconstructing conceptual whole-plants would allow the Loch Humphrey Burn pteridosperms in particular to be explored in the wider context of broad-brush morphological phylogenetics e.g., . It woulThe Mississippian stratigraphy of the Scottish Midland Valley is in turn critical to solving the outstanding controversies in recent tectonic models. To quote