Aquatic Ferrous Solutions of Prebiotic Mineral Salts as Strong UV Protectants and Possible Loci of Life Origin.

Liposomes are lipid-bilayer vesicles that spontaneously self-assemble from fatty acids (or other amphiphiles) in water by encapsulating surrounding aqueous media. After British scientist Alec Bangham described this phenomenon in the early 1960s, they became a prominent participant in the hypotheses on life origin, particularly in the Lipid World model. A novel scenario of self-sustained Darwinian liposome evolution is based on ever-present natural phenomena of cyclic day/night solar UV radiation and gravitational submersion of liposomes in the Archean aqueous media. One of the assumptions of the hypothesis is the UV-shielding ability of the Archean waters that could protect the submerged liposomes from the damaging solar UV radiation. To corroborate the idea, we measured UV absorption in aquatic solutions of several ferrous mineral salts assumed to be present in Archean pools. Single-agent solutions of simple salts such as FeCl2-iron dichloride, FeCl3-iron trichoride, Fe(NO3)3-ferric nitride, NH4Fe(SO4)2-ferric ammonium sulfate, and (NH4)5[Fe(C6H4O7)2]-ferric ammonium citrate were tested. These direct measurements of UV light absorption supplement and reinforce the proposed hypothesis.

Exploring the Lipid World Hypothesis: A Novel Scenario of Self-Sustained Darwinian Evolution of the Liposomes.

According to the Lipid World hypothesis, life on Earth originated with the emergence of amphiphilic assemblies in the form of lipid micelles and vesicles (liposomes). However, the mechanism of appearance of the information molecules (ribozymes/RNA) accompanying that process, considered obligatory for Darwinian evolution, is unclear. We propose a novel scenario of self-sustained Darwinian evolution of the liposomes driven by ever-present natural phenomena: solar UV radiation, day/night cycle, gravity, and the formation of liposomes in an aqueous media. The central tenet of this scenario is the liposomes' encapsulation of the heavy solutes, followed by their gravitational submerging in the water. The submerged liposomes, being protected from the damaging UV radiation, acquire the longevity necessary for autocatalytic replication of amphiphiles, their mutation, and the selection of those amphiphilic assemblies that provide the greatest membrane stability. These two sets of adaptive compositional information (heavy content and amphiphilic assemblies design) generate a population of liposomes with self-replication/reproduction properties, which are amendable to mutation, inheritance, and selection, thereby establishing Darwinian progression. Temporary and spatial expansion of this liposomal population will provide the basis for the next evolutionary step-a transition of accidentally entrapped RNA precursor molecules into complex functional molecules, such as ribozymes/RNA.

Short-course neoadjuvant in situ vaccination for murine melanoma.

BACKGROUND: Surgical resection remains an important component of multimodality treatment for most solid tumors. Neoadjuvant immunotherapy has several potential advantages, including in-situ tumor vaccination and pathologic assessment of response in the surgical specimen. We previously described an in-situ tumor vaccination strategy in melanoma using local radiation (RT) and an intratumoral injection of tumor-specific anti-GD2 immunocytokine (IT-IC). Here we tested whether neoadjuvant in-situ tumor vaccination using anti-GD2 immunocytokine and surgical resection, without RT, could generate immunologic memory capable of preventing recurrence or distant metastasis. METHODS: Mice bearing GD2 expressing B78 melanoma tumors were treated with neoadjuvant radiation, IT-IC, or combined RT + IT-IC. Surgical resection was performed following neoadjuvant immunotherapy. Immune infiltrate was assessed in the resection specimens. Mice were rechallenged with either B78 contralateral flank tumors or pulmonary seeding of non-GD2 expressing B16 melanoma metastasis induced experimentally. Rejection of rechallenge in mice treated with the various treatment regimens was considered evidence of immunologic memory. RESULTS: Neoadjuvant IT-IC and surgical resection resulted in increased CD8 T cell infiltration, a higher CD8:regulatory T cell ratio, and immunologic memory against contralateral flank rechallenge. The timing of resection did not significantly impact the development of memory, which was present as early as the day of surgery. There was less local wound toxicity with neoadjuvant IT-IC compared with neoadjuvant RT +IT IC. Neoadjuvant IT-IC and resection resulted in the rejection of B16 lung metastasis in a CD4 T cell dependent manner. CONCLUSIONS: Neoadjuvant IT-IC generates immunologic memory capable of preventing distant metastasis despite limited efficacy against large primary melanoma tumors. By combining neoadjuvant tumor vaccination and surgery, the toxicity of local RT was avoided. These preclinical data support further investigation regarding the use of neoadjuvant IT-IC in patients with melanoma at high risk for occult distant disease.

Etiopathogenesis of adolescent idiopathic scoliosis: Review of the literature and new epigenetic hypothesis on altered neural crest cells migration in early embryogenesis as the key event.

Adolescent idiopathic scoliosis (AIS) affects 2-3% of children. Numerous hypotheses on etiologic/causal factors of AIS were investigated, but all failed to identify therapeutic targets and hence failed to offer a cure. Therefore, currently there are only two options to minimize morbidity of the patients suffering AIS: bracing and spinal surgery. From the beginning of 1960th, spinal surgery, both fusion and rod placement, became the standard of management for progressive adolescent idiopathic spine deformity. However, spinal surgery is often associated with complications. These circumstances motivate AIS scientific community to continue the search for new etiologic and causal factors of AIS. While the role of the genetic factors in AIS pathogenesis was investigated intensively and universally recognized, these studies failed to nominate mutation of a particular gene or genes combination responsible for AIS development. More recently epigenetic factors were suggested to play causal role in AIS pathogenesis. Sharing this new approach, we investigated scoliotic vertebral growth plates removed during vertebral fusion (anterior surgery) for AIS correction. In recent publications we showed that cells from the convex side of human scoliotic deformities undergo normal chondrogenic/osteogenic differentiation, while cells from the concave side acquire a neuronal phenotype. Based on these facts we hypothesized that altered neural crest cell migration in early embryogenesis can be the etiological factor of AIS. In particular, we suggested that neural crest cells failed to migrate through the anterior half of somites and became deposited in sclerotome, which in turn produced chondrogenic/osteogenic-insufficient vertebral growth plates. To test this hypothesis we conducted experiments on chicken embryos with arrest neural crest cell migration by inhibiting expression of Paired-box 3 (Pax3) gene, a known enhancer and promoter of neural crest cells migration and differentiation. The results showed that chicken embryos treated with Pax3 siRNA (microinjection into the neural tube, 44 h post-fertilization) progressively developed scoliotic deformity during maturation. Therefore, this analysis suggests that although adolescent idiopathic scoliosis manifests in children around puberty, the real onset of the disease is of epigenetic nature and takes place in early embryogenesis and involves altered neural crest cells migration. If these results confirmed and further elaborated, the hypothesis may shed new light on the etiology and pathogenesis of AIS.

Depth of tumor implantation affects response to in situ vaccination in a syngeneic murine melanoma model.

An important component of research using animal models is ensuring rigor and reproducibility. This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy. Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC). Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically 'cold' tumors, has not been well studied. The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation. We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank. When tumors reached 190-230 mm3, they were grouped into a 'wave' and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine). Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia. Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors. SC tumors reached the target tumor volume significantly faster compared with ID tumors. Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not. Further, the 'mobile' or 'fixed' phenotype of tumors predicted response to therapy, regardless of intended implantation depth. These findings were then extended to additional immunotherapy regimens in four separate tumor models. These data indicate that the physical 'fixed' versus 'mobile' characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment. Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.

Pattern of organ remodeling in chronic non-communicable diseases is due to endogenous regulations and falls under the category of Kauffman's self-organization: A case of arterial neointimal pathology.

Clinical diagnosis is based on analysis of pathologic findings that may result in perceived patterns. The same is true for diagnostic pathology: Pattern analysis is a foundation of the histopathology-based diagnostic system and, in conjunction with clinical and laboratory findings, forms a basis for the classification of diseases. Any histopathology diagnosis is based on the explicit assumption that the same diseased condition should result in formation of the same (or highly similar) morphologic patterns in different individuals; it is a standard approach in microscopic pathology, including that of non-communicable chronic diseases with organ remodeling. During fifty years of examining diseased tissues under microscopy, I keep asking the same question: Why is a similarity of patterns expected for chronic organ remodeling? For infection diseases, xenobiotic toxicity and deficiencies forming an identical pathologic pattern in different individuals is understandable and logical: The same infection, xenobiotic, or deficiency strikes the same target, which results in identical pathology. The same is true for Mendelian diseases: The same mutations lead to the same altered gene expressions and the same pathologic pattern. But why does this regularity hold true for chronic diseases with organ remodeling? Presumable causes (or risk factors) for a particular chronic disease differ in magnitude and duration between individuals, which should result in various series of transformations. Yet, mysteriously enough, pathological remodeling in a particular chronic disease always falls into a main dominating pattern, perpetuating and progressing in a similar fashion in different patients. Furthermore, some chronic diseases of different etiologies and dissimilar causes/risk factors manifest as identical or highly similar patterns of pathologic remodeling. HYPOTHESIS: I hypothesize that regulations governing a particular organ's chronic remodeling were selected in evolution as the safest response to various insults and physiologic stress conditions. This hypothesis implies that regulations directing diseased chronic remodeling always preexist but normally are controlled; this control can be disrupted by a diverse range of non-specific signals, liberating the pathway for identical pathologic remodeling. This hypothesis was tested in an analysis of arterial neointimal formation, the identical pathology occurring in different diseases and pathological conditions: graft vascular disease in organ transplantation, in-stent restenosis, peripheral arterial diseases, idiopathic intimal hyperplasia, Kawasaki disease, coronary atherosclerosis and as reaction to drugs. The hypothesis suggests that arterial intimal cells are poised between only two alternative pathways: the pathway with controlled intimal cell proliferation or the pathway where such control is disrupted, ultimately leading to the progressive neointimal pathology. By this property the arterial neointimal formation constitutes a special case of Kauffman's self-organization. This new hypothesis gives a parsimonious explanation for identical pathological patterns of arterial remodeling (neointimal formation), which occurs in diseases of different etiologies and due to dissimilar causes/risk factors, or without any etiology and causes/risk factors at all. This new hypothesis also suggests that regulation facilitating intimal cell proliferation cannot be overwritten or annulled because this feature is vital for arterial differentiation, cell renewal, and integrity. This hypothesis suggests that studying numerous, and likely interchangeable, non-specific signals that disrupt regulation controlling intimal cell proliferation is unproductive; instead, a study of the controlling regulation(s) itself should be a priority of our research.

Development of an RNAi therapeutic for alpha-1-antitrypsin liver disease.

The autosomal codominant genetic disorder alpha-1 antitrypsin (AAT) deficiency (AATD) causes pulmonary and liver disease. Individuals homozygous for the mutant Z allele accumulate polymers of Z-AAT protein in hepatocytes, where AAT is primarily produced. This accumulation causes endoplasmic reticulum (ER) stress, oxidative stress, damage to mitochondria, and inflammation, leading to fibrosis, cirrhosis, and hepatocellular carcinoma. The magnitude of AAT reduction and duration of response from first-generation intravenously administered RNA interference (RNAi) therapeutic ARC-AAT and then with next-generation subcutaneously administered ARO-AAT were assessed by measuring AAT protein in serum of the PiZ transgenic mouse model and human volunteers. The impact of Z-AAT reduction by RNAi on liver disease phenotypes was evaluated in PiZ mice by measuring polymeric Z-AAT in the liver; expression of genes associated with fibrosis, autophagy, apoptosis, and redox regulation; inflammation; Z-AAT globule parameters; and tumor formation. Ultrastructure of the ER, mitochondria, and autophagosomes in hepatocytes was evaluated by electron microscopy. In mice, sustained RNAi treatment reduced hepatic Z-AAT polymer, restored ER and mitochondrial health, normalized expression of disease-associated genes, reduced inflammation, and prevented tumor formation. RNAi therapy holds promise for the treatment of patients with AATD-associated liver disease. ARO-AAT is currently in phase II/III clinical trials.

Modeling multi-needle injection into solid tumor.

The discovery of mechanisms by which the cancer cells avoid the host immune attack (immune checkpoints) as well the capability of the monoclonal antibodies (mAbs) to blockade the checkpoint proteins on cancer and tumor-infiltrating cells (CTLA-4, PD-1, and PD-L1) promised new breakthroughs in the cure of cancer. After these mechanisms of cancer escaping the host immunity were undoubtedly confirmed in numerous experimental and clinical studies, the FDA approval of CTLA-4 and PD-1/PD-L1 mAbs for systemic treatment thought to revolutionize the outcome of cancer treatment. However, as of today, the anticipated curative effect of anti-CTLA-4 and PD-1/PD-L1 mAb treatments has been observed only in a small population of patients. In addition, systemic administration of mAbs in clinics has been found associated with new toxicity profiles, sometimes very severe. The main obstacle that hinders the mAbs therapy appears to be the inability of delivering mAbs to a sufficient number of cancer cells and tumor infiltrating cells. As an alternative to the systemic administration (or as a complement to it), local intratumoral delivery of mAbs has been anticipated to resolve that issue. However, unlike the systemic mAbs administration, for which formidable but surmountable obstacles (big size of mAbs ~150 kD, high interstitial fluid pressure in solid tumors, etc.) have been known to hamper mAbs delivery to cancer and tumor-infiltrating cells, the lack of effects of intratumoral mAbs administration remains completely incomprehensible and needs a new theoretical reconsideration that we have attempted in our analysis. It can be suggested that the limited benefits of the intratumoral mAbs administration appeared to be rooted in the same problem that hindered the effects of systemic mAbs administration: the inability to reach a sufficient number of cancer cells and tumor-infiltrating cells. We hypothesize that the core of the problem stems from the fact that the single-needle intratumoral injection forms a very localized, jet-like distribution of the drug (mAbs) that constitutes only a small fraction of the total volume of the tumor. In this light we are re-evaluating the theoretical reasonableness of the single-needle intratumoral injection approach. We propose that multi-needle injection will circumvent this limitation and for that we analyze the behavior of an injectant in tissues using different configurations of the injection needles. To accomplish this goal, we created a model of injectant distribution in a solid tissue based on the traditional technique of single-needle injection and then extended that model to a case of simultaneous multi-needle injection. To develop the model of drug delivery and transport in biological tissues, we followed a frequently used approach of modeling the diffusive transport of liquid through a porous media using the Darcy's law that relates the flow velocity, the pressure gradient, and the tissue permeability. The analysis demonstrates that a multi-needle injection setup provides a significantly more widespread and homogeneous injectant distribution within a solid tumor than that for a single needle injection for the same tumor size. Adding separate draining needles can further improve the delivery of injectant to cancer and tumor-infiltrating cells.

A hypothesis on paradoxical privileged portal vein metastasis of hepatocellular carcinoma. Can organ evolution shed light on patterns of human pathology, and vice versa?

Unlike other carcinomas, hepatocellular carcinoma (HCC) metastasizes to distant organs relatively rarely. In contrast, it routinely metastasizes to liver vasculature/liver, affecting portal veins 3-10 times more often than hepatic veins. This portal metastatic predominance is traditionally rationalized within the model of a reverse portal flow, due to accompanying liver cirrhosis. However, this intuitive model is not coherent with facts: 1) reverse portal flow occurs in fewer than 10% of cirrhotic patients, while portal metastasis occurs in 30-100% of HCC cases, and 2) portal vein prevalence of HCC metastasis is also characteristic of HCC in non-cirrhotic livers. Therefore, we must assume that the route for HCC metastatic dissemination is the same as for other carcinomas: systemic dissemination via the draining vessel, i.e., via the hepatic vein. In this light, portal prevalence versus hepatic vein of HCC metastasis appears as a puzzling pattern, particularly in cases when portal HCC metastases have appeared as the sole manifestation of HCC. Considering that other GI carcinomas (colorectal, pancreatic, gastric and small bowel) invariably disseminate via portal vein, but very rarely form portal metastasis, portal prevalence of HCC metastasis appears as a paradox. However, nature does not contradict itself; it is rather our wrong assumptions that create paradoxes. The 'portal paradox' becomes a logical event within the hypothesis that the formation of the unique portal venous system preceded the appearance of liver in evolution of chordates. The analysis suggests that the appearance of the portal venous system, supplying hormones and growth factors of pancreatic family, which includes insulin, glucagon, somatostatin, and pancreatic polypeptide (HGFPF) to midgut diverticulum in the early evolution of chordates (in an Amphioxus-like ancestral animal), promoted differentiation of enterocytes into hepatocytes and their further evolution to the liver of vertebrates. These promotional-dependent interactions are conserved in the vertebrate lineage. I hypothesize that selective homing and proliferation of malignant hepatocytes (i.e., HCC cells) in the portal vein environment are due to a uniquely high concentration of HGFPF in portal blood. HGFPF are also necessary for liver function and renewal and are significantly extracted by hepatocytes from passing blood, creating a concentration gradient of HGFPF between the portal blood and hepatic vein outflow, making post-liver vasculature and remote organs less favorable spaces for HCC growth. It also suggested that the portal vein environment (i.e., HGFPF) promotes the differentiation of more aggressive HCC clones from already-seeded portal metastases, explaining the worse outcome of HCC with the portal metastatic pattern. The analysis also offers new hypothesis on the phylogenetic origin of the hepatic diverticulum of cephalochordates, with certain implications for the modeling of the chordate phylogeny.