The Mechanism of Dual Responsiveness in Muscle Fibers of the Grasshopper Romalea microptera

Dually innervated Romalea muscle fibers which respond differently to stimulation of their fast and slow axons are excited by intracellularly applied depolarizing stimuli. The responses, though spike-like in appearance, are graded in amplitude depending upon the strength of the stimuli and do not exceed about 30 mv. in height. In other respects, however, these graded responses possess properties that are characteristic of electrically excitable activity: vanishingly brief latency; refractoriness; a post-spike undershoot. They are blocked by hyperpolarizing the fiber membrane; respond repetitively to prolonged depolarization, and are subject to depolarizing inactivation. As graded activity, these responses propagate decrementally. The fast and slow axons of the dually responsive muscle fibers initiate respectively large and small postsynaptic potentials (p.s.p.'s) in the muscle fiber. These responses possess properties that characterize electrically inexcitable depolarizing activity. They are augmented by hyperpolarization and diminished by depolarization. Their latency is independent of the membrane potential. They have no refractory period, thus being capable of summation. The fast p.s.p. evokes a considerable or maximal electrically excitable response. The combination, which resembles a spike, leads to a twitch-like contraction of the muscle fiber. The individual slow p.s.p.'s elicit no or only little electrically excitable responses, and they evoke slower smaller contractile responses. The functional aspects of dual responsiveness and the several aspects of the theoretical importance of the gradedly responsive, electrically excitable component are discussed.     

Inhibitory Miniature Potentials in the Stretch Receptor Neurons of Crayfish

Intracellular microelectrodes inserted into the soma of crayfish stretch receptor neurons record frequent fluctuations of the membrane potential. Time course, amplitude, and interval distribution indicate that they are miniature potentials. At the average resting potential the polarity of the miniature potentials depends on the anion used in the microelectrode: KCl electrodes record depolarizing, K citrate or K₂SO₄ electrodes, hyperpolarizing miniature potentials. The inhibitory postsynaptic potentials (i.p.s.p.'s) show a similar polarity change. The reversal potentials of i.p.s.p.'s and miniature potentials are equal and within 10 mv of the resting potential, more negative with K citrate (or K₂SO₄), less negative with KCl electrodes. Reversal can be accomplished by changing the membrane potential by stretching or by current passing. Injection of Cl^- into the soma or replacement of external Cl by propionate results in an abrupt increase of the amplitude of the miniature potentials lasting for several minutes. The miniature potentials like the i.p.s.p.'s are reversibly abolished by the application of picrotoxin and γ-aminobutyric acid. They are not affected by tetrodotoxin, nor by acetylocholine, eserine, or atropine. It is concluded that the miniature potentials represent a spontaneous quantal release of transmitter substance from inhibitory nerve terminals, and that the transmitter substance predominantly increases the Cl^- permeability of the postsynaptic membrane. The effect of the spontaneously released transmitter on the behavior of the receptor neuron is considerable. The membrane conductance is increased by up to 36% and the excitability is correspondingly depressed.     

Regulation of PI(3)K/Akt signalling and cellular transformation by inositol polyphosphate 4‐phosphatase‐1

Akt is a crucial phosphoinositide 3-kinase (PI(3)K) effector that regulates cell proliferation and survival. PI(3)K-generated signals, PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, direct Akt plasma membrane engagement. Pathological Akt plasma membrane association promotes oncogenesis. PtdIns(3,4)P₂ is degraded by inositol polyphosphate 4-phosphatase-1 (4-ptase-1) forming PtdIns(3)P; however, the role of 4-ptase-1 in regulating the activation and function of Akt is unclear. In mouse embryonic fibroblasts lacking 4-ptase-1 (^−/−MEFs), the Akt-pleckstrin homology (PH) domain was constitutively membrane-associated both in serum-starved and agonist-stimulated cells, in contrast to ^+/+MEFs, in which it was detected only at the plasma membrane following serum stimulation. Epidermal growth factor (EGF) stimulation resulted in increased Ser⁴⁷³ and Thr³⁰⁸-Akt phosphorylation and activation of Akt-dependent signalling in ^−/−MEFs, relative to ^+/+MEFs. Significantly, loss of 4-ptase-1 resulted in increased cell proliferation and decreased apoptosis. SV40-transformed ^−/−MEFs showed increased anchorage-independent cell growth and formed tumours in nude mice. This study provides the first evidence, to our knowledge, that 4-ptase-1 controls the activation of Akt and thereby cell proliferation, survival and tumorigenesis.     

Potassium homeostasis in the nervous system of cephalopods and crustacea

1. Previous work has shown that nerve activity is associated with a significant release of potassium in the vicinity of the axonal membrane. Several mechanisms are normally present which reduce K+ accumulation in the extra-axonal space. 2. In intact connectives of the crayfish, Procambarus clarkii, repetitive stimulation of the giant axons was associated with an apparent hyperpolarization measured by an interstitial microelectrode, which most probably corresponds to depolarization of the inner face of the perineurial cells by K+ ions leaving the axons. 3. In desheathed connectives of the crayfish, potassium accumulated during long depolarizing voltage-clamp pulses but cleared away very quickly at the end of the pulse. 4. In the small squid, Alloteuthis subulata, repetitive stimulation of giant axons in situ in fresh and well-perfused animals did not result in a large decrease in the positive after potential (undershoot), reflecting the absence of potassium accumulation. A similar absence of accumulation was observed in vitro for carefully and freshly dissected isolated axons from live squids. 5. In both cases, deterioration of the physiological state of the axon was accompanied by a significant potassium accumulation. Potassium accumulation could also be reversibly enhanced by decreasing the osmotic pressure of the bathing medium, whereas hyperosmotic solutions had the opposite effect. These results are compatible with the idea that Schwann cells around the axon play a key role in K+ homeostasis. 6. Experiments on giant axons of the large squid species, Loligo forbesi confirmed the observations made on Alloteuthis in that fresh preparations exhibited little potassium accumulation. Under voltage-clamp conditions, 10 ms depolarizing pulses to various potential levels did not induce any accumulation in these preparations as reflected by the outward tail current. Large accumulation was observed in older axons under similar experimental conditions. 7. A large peri-axonal space associated with healthy glial cells appears to be a prerequisite for efficient K+ homeostasis in both crayfish and squid. Other mechanisms involving specific transport mechanisms across axonal and glial membranes are also likely to be involved.     

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose

Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice.

Methods

The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8 ^+/− mice expressing ß-galactosidase. Aged Mfge8 ^+/− and Mfge8 ^−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV.

Results

rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8^−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch''s membrane (BM) was slightly but significantly thicker in Mfge8^−/− mice as compared to controls.

Conclusions

Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8^−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.     

ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA

Background

Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.

Methodology/Principal Findings

Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR^−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR^−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR^−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.

Conclusions

proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.     

HMGB‐1 induces c‐kit+ cell microvascular rolling and adhesion via both toll‐like receptor‐2 and toll‐like receptor‐4 of endothelial cells

High-mobility group box 1 (HMGB-1) is a strong chemo-attractive signal for both inflammatory and stem cells. The aim of this study is to evaluate the mechanisms regulating HMGB-1–mediated adhesion and rolling of c-kit⁺ cells and assess whether toll-like receptor-2 (TLR-2) and toll-like receptor-4 (TLR-4) of endothelial cells or c-kit⁺ cells are implicated in the activation of downstream migration signals to peripheral c-kit⁺ cells. Effects of HMGB-1 on the c-kit⁺ cells/endothelial interaction were evaluated by a cremaster muscle model in wild-type (WT), TLR-2 (−/−) and Tlr4 (LPS-del) mice. The mRNA and protein expression levels of endothelial nitric oxide synthase were determined by quantitative real-time PCR and immunofluorescence staining. Induction of crucial adhesion molecules for rolling and adhesion of stem cells and leukocytes were monitored in vivo and in vitro. Following local HMGB-1 administration, a significant increase in cell rolling was detected (32.4 ± 7.1% in ‘WT’ versus 9.9 ± 3.2% in ‘control’, P < 0.05). The number of firmly adherent c-kit⁺ cells was more than 13-fold higher than that of the control group (14.6 ± 5.1 cells/mm² in ‘WT’ versus 1.1 ± 1.0 cells/mm² in ‘control’, P < 0.05). In knockout animals, the fraction of rolling cells did not differ significantly from control levels. Firm endothelial adhesion was significantly reduced in TLR-2 (−/−) and Tlr4 (LPS-del) mice compared to WT mice (1.5 ± 1.4 cells/mm² in ‘TLR-2 (−/−)’ and 2.4 ± 1.4 cells/mm² in ‘Tlr4 (LPS-del)’ versus 14.6 ± 5.1 cells/mm² in ‘WT’, P < 0.05). TLR-2 (−/−) and Tlr4 (LPS-del) stem cells in WT mice did not show significant reduction in rolling and adhesion compared to WT cells. HMGB-1 mediates c-kit⁺ cell recruitment via endothelial TLR-2 and TLR-4.     

Impaired Glucose Tolerance in a Mouse Model of Sidt2 Deficiency

Sidt2 was identified as a novel integral lysosomal membrane protein recently. We generated global Sidt2 knockout mice by gene targeting. These mice have a comparatively higher random and fasting glucose concentration. Intraperitoneal and oral glucose tolerance tests in Sidt2 knockout mice indicated glucose intolerance and decreased serum insulin level. Notably, the Sidt2^−/− mice had hypertrophic islets compared with control mice. By Western blot and immunofluorescence, Sidt2^−/− mouse islets were shown to have increased insulin protein, which actually contained more insulin secretory granules than their controls, demonstrated by electromicroscopy. Consistent with the in vivo study, isolated islet culture from the Sidt2^−/− mice produced less insulin when stimulated by a high concentration of glucose or a depolarizing concentration of KCl. Under electromicroscope less empty vesicles and more mature ones in Sidt2^−/− mice islets were observed, supporting impaired insulin secretory granule release. In conclusion, Sidt2 may play a critical role in the regulation of mouse insulin secretory granule secretion.     

Association of JAG1 with Bone Mineral Density and Osteoporotic Fractures: A Genome-wide Association Study and Follow-up Replication Studies

Bone mineral density (BMD), a diagnostic parameter for osteoporosis and a clinical predictor of fracture, is a polygenic trait with high heritability. To identify genetic variants that influence BMD in different ethnic groups, we performed a genome-wide association study (GWAS) on 800 unrelated Southern Chinese women with extreme BMD and carried out follow-up replication studies in six independent study populations of European descent and Asian populations including 18,098 subjects. In the meta-analysis, rs2273061 of the Jagged1 (JAG1) gene was associated with high BMD (p = 5.27 × 10⁻⁸ for lumbar spine [LS] and p = 4.15 × 10⁻⁵ for femoral neck [FN], n = 18,898). This SNP was further found to be associated with the low risk of osteoporotic fracture (p = 0.009, OR = 0.7, 95% CI 0.57–0.93, n = 1881). Region-wide and haplotype analysis showed that the strongest association evidence was from the linkage disequilibrium block 5, which included rs2273061 of the JAG1 gene (p = 8.52 × 10⁻⁹ for LS and 3.47 × 10⁻⁵ at FN). To assess the function of identified variants, an electrophoretic mobility shift assay demonstrated the binding of c-Myc to the “G” but not “A” allele of rs2273061. A mRNA expression study in both human bone-derived cells and peripheral blood mononuclear cells confirmed association of the high BMD-related allele G of rs2273061 with higher JAG1 expression. Our results identify the JAG1 gene as a candidate for BMD regulation in different ethnic groups, and it is a potential key factor for fracture pathogenesis.     

Effect of ionic contents in saline on depolarizing afterpotential induced by phenothrin and methoxychlor

1. The depolarizing afterpotential induced by phenothrin and methoxychlor in crayfish giant axons was measured intracellularly. 2. Changes in the K+ ion concentration did not affect the depolarizing afterpotential when evaluated as the membrane potential. 3. Addition of tetrodotoxin or replacement of the Cl- ions by OAc- ions or of the Na+ ions by choline ions reduced the depolarizing afterpotential and the action potential. 4. A ratio of the reduction of the depolarizing afterpotential in saline that contained OAc- ions to that of the action potential was similar to that measured in the saline containing tetrodotoxin. 5. With the increase in the choline concentration, the relative value of the depolarizing afterpotential to that measured before the replacement decreased almost linearly with that of the decrease in the action potential.     

In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish

Background

Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/Principal Findings

Analysis of the zebrafish shh ^−/− mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh ^−/− mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh ^−/− mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh ^−/− mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 ^−/− shh ^−/− mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions

Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.     

Migration and differentiation of transplanted enteric neural crest-derived cells in murine model of Hirschsprung’s disease

Stem cell therapy offers the potential of rebuilding the enteric nervous system (ENS) in the aganglionic bowel of patients with Hirschsprung’s disease. P0-Cre/Floxed-EGFP mice in which neural crest-derived cells express EGFP were used to obtain ENS stem/progenitor cells. ENS stem/progenitor cells were transplanted into the bowel of Ret^−/− mouse, an animal model of Hirschsprung’s disease. Immunohistochemical analysis was performed to determine whether grafted cells gave rise to neurons in the recipient bowel. EGFP expressing neural crest-derived cells accounted for 7.01 ± 2.52 % of total cells of gastrointestinal tract. ENS stem/progenitor cells were isolated using flow cytometry and expanded as neurosphere-like bodies (NLBs) in a serum-free culture condition. Some cells in NLBs expressed neural crest markers, p75 and Sox10 and neural stem/progenitor cells markers, Nestin and Musashi1. Multipotency of isolated ENS stem/progenitor cells was determined as they differentiated into neurons, glial cells, and myofibloblasts in culture. When co-cultured with explants of hindgut of Ret^−/− mice, ENS stem/progenitor cells migrated into the aganglionic bowel and gave rise to neurons. ENS stem/progenitor cells used in this study appear to be clinically relevant donor cells in cell therapy to treat Hirschsprung’s disease capable of colonizing the affected bowel and giving rise to neurons.     

Studies of the Uptake of Nitrate in Barley : IV. Electrophysiology

Transmembrane electrical potential differences (Δψ) of epidermal and cortical cells were measured in intact roots of barley (Hordeum vulgare L. cv Klondike). The effects of exogenous NO₃⁻ on Δψ (in the concentration range from 100 micromolar to 20 millimolar) were investigated to probe the mechanisms of nitrate uptake by the high-affinity (HATS) and low-affinity (LATS) transport systems for NO₃⁻ uptake. Both transport systems caused depolarization of Δψ, demonstrating that the LATS (like the HATS) for NO₃⁻ uptake is probably mediated by an electrogenic cation (H⁺?) cotransport system. Membrane depolarization by the HATS was “inducible” by NO₃⁻, and saturable with respect to exogenous [NO₃⁻]. By contrast, depolarization by the LATS was constitutive, and first-order in response to external [NO₃⁻]. H⁺ fluxes, measured in 200 micromolar and in 5 millimolar Ca(NO₃)₂ solutions, failed to alkalinize external media as anticipated for a 2 H⁺:1 NO₃⁻ symport. However, switching from K₂SO₄ solutions (which were strongly acidifying) to KNO₃ solutions at the same K⁺ concentration caused marked reductions in H⁺ efflux. These observations are consistent with NO₃⁻ uptake by the HATS and the LATS via 2 H⁺:1 NO₃⁻ symports. These observations establish that the HATS for nitrate uptake by barley roots is essentially similar to those reported for Lemna and Zea mays by earlier workers. There are, nevertheless, distinct differences between barley and corn in their quantitative responses to external NO₃⁻.     

HIV-1 infection abrogates CD8+ T cell mitogen-activated protein kinase signaling responses

Mitogen-activated protein kinase (MAPK) signaling pathways are dynamic and sensitive regulators of T cell function and differentiation. Altered MAPK signaling has been associated with the inflammatory and autoimmune diseases lupus and arthritis and with some pathogenic viral infections. HIV-1 infection is characterized by chronic immune inflammation, aberrantly heightened CD8⁺ T cell activation levels, and altered T cell function. The relationship between MAPK pathway function, HIV-1-induced activation (CD38 and HLA-DR), and exhaustion (Tim-3) markers in circulating CD8⁺ T cells remains unknown. Phosphorylation of the MAPK effector proteins ERK and p38 was examined by “phosflow” flow cytometry in 79 recently HIV-1-infected, antiretroviral-treatment-naïve adults and 21 risk-matched HIV-1-negative controls. We identified a subset of CD8⁺ T cells refractory to phorbol 12-myristate 13-acetate plus ionomycin-induced ERK1/2 phosphorylation (referred to as p-ERK1/2-refractory cells) that was greatly expanded in HIV-1-infected adults. The CD8⁺ p-ERK1/2-refractory cells were highly activated (CD38⁺ HLA-DR⁺) but not exhausted (Tim-3 negative), tended to have low CD8 expression, and were enriched in intermediate and late transitional memory states of differentiation (CD45RA⁻ CD28⁻ CD27^+/−). Targeting MAPK pathways to restore ERK1/2 signaling may normalize immune inflammation levels and restore CD8⁺ T cell function during HIV-1 infection.     

A non‐redundant role for MKP5 in limiting ROS production and preventing LPS‐induced vascular injury

There are at least 11 mitogen-activated protein kinase (MAPK) phosphatases (MKPs) and only 3 major groups of MAPKs, raising the question of whether these phosphatases have non-redundant functions in vivo. Using a modified mouse model of local Shwartzman reaction, we found that deletion of the MKP5 gene, but not the MKP1 gene, led to robust and accelerated vascular inflammatory responses to a single dose of LPS injection. Depletion of neutrophils significantly reduced the vascular injury in Mkp5^−/− mice, whereas adoptive transfer of Mkp5^−/− neutrophils replicated the LPS-induced skin lesions in wild-type recipients. Neutrophils isolated from Mkp5^−/− mice exhibited augmented p38 MAPK activation and increased superoxide generation on activation. The p38 MAPK inhibitor, SB203580, significantly reduced p47^phox phosphorylation and diminished superoxide production in neutrophils. p38 MAPK phosphorylated mouse p47^phox, and deletion of the p47^phox gene ablated the LPS-induced vascular injury in Mkp5^−/− mice. Collectively, these results show an earlier unrecognized and non-redundant function of MKP5 in restraining p38 MAPK-mediated neutrophil oxidant production, thereby preventing LPS-induced vascular injury.     

Wild-type p53-induced phosphatase 1 (Wip1) forestalls cellular premature senescence at physiological oxygen levels by regulating DNA damage response signaling during DNA replication

Wip1 (protein phosphatase Mg²⁺/Mn²⁺-dependent 1D, Ppm1d) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Ppm1d^−/− mouse embryonic fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions; however, little is known regarding the role of Wip1 in regulating cellular senescence. In this study, we found that even at a representative physiological concentration of 3% O₂, Ppm1d^−/− MEFs underwent premature cellular senescence that depended on the functional activation of p53. Interestingly, Ppm1d^−/− MEFs showed increased H2AX phosphorylation levels without increased levels of reactive oxygen species (ROS) or DNA base damage compared with wild-type (Wt) MEFs, suggesting a decreased threshold for DDR activation or sustained DDR activation during recovery. Notably, the increased H2AX phosphorylation levels observed in Ppm1d^−/− MEFs were primarily associated with S-phase cells and predominantly dependent on the activation of ATM. Moreover, these same phenotypes were observed when Wt and Ppm1d^−/− MEFs were either transiently or chronically exposed to low levels of agents that induce replication-mediated double-stranded breaks. These findings suggest that Wip1 prevents the induction of cellular senescence at physiological oxygen levels by attenuating DDR signaling in response to endogenous double-stranded breaks that form during DNA replication.     

KSR Int'l Co. v. Teleflex, Inc.: no obvious changes for the biotechnology market

With the advent of molecular biology, genomics, and proteomics, the intersection between science and law has become increasingly significant. In addition to the ethical and legal concerns surrounding the collection, storage, and use of genomic data, patent disputes for new biotechnologies are quickly becoming part of mainstream business discussions. Under current patent law, new technologies cannot be patented if they are “obvious” changes to an existing patent. The definition of “obvious,” therefore, has a huge impact on determining whether a patent is granted. For example, are modifications to microarray protocols, popular in diagnostic medicine, considered “obvious” improvements of previous products? Also, inventions that are readily apparent now may not have been obvious when discovered. Polymerase chain reaction, or PCR, is now a common component of every biologist’s toolbox and seems like an obvious invention, though it clearly was not in 1983. Thus, there is also a temporal component that complicates the interpretation of an invention’s obviousness. The following article discusses how a recent Supreme Court decision has altered the definition of “obviousness” in patent disputes. By examining how the obviousness standard has changed, the article illuminates how legal definitions that seem wholly unrelated to biology or medicine could still potentially have enormous effects on these fieldsJust what is obvious or not is a question that has provoked substantial litigation in the Federal Circuit, the appellate court with special jurisdiction over patent law disputes. Under U.S. patent law, an inventor may not obtain a patent, which protects his invention from infringement by others, if the differences between the subject matter sought to be patented and the prior art are such that “the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill” in the patent’s subject matter area [1]. However, what was “obvious” at the time of invention to a person of ordinary skill is hardly clear and is, in effect, a legal fiction designed to approximate objectivity. As illustrated by Chief Justice John Roberts of the Supreme Court in a moment of levity, “Who do you get to ... tell you something’s not obvious … the least insightful person you can find?” [2] Despite the apparent objectivity provided by a “person of ordinary skill” obviousness standard, the difficulty lies in that such a standard is still susceptible to multiple interpretations, depending on the point of view and knowledge ascribed to the “ordinary person.” As such, how obviousness is defined and interpreted by the courts will have important implications on biotechnology patents and the biotechnology business.The issue of obviousness arose in April 2007 when the Supreme Court handed down its decision in KSR Int’l Co. v. Teleflex, Inc. [3] The facts of the case were anything but glamorous; in the suit, Teleflex, a manufacturer of adjustable pedal systems for automobiles, sued KSR, its rival, for infringement of its patent, which “describe[d] a mechanism for combining an electronic sensor with an adjustable automobile pedal so that the pedal’s position can be transmitted to a computer that controls the throttle in the vehicle’s engine.” [4] Teleflex believed that KSR’s new pedal design was too similar to its own patented design and therefore infringed upon it [5]. In defense, KSR argued that Teleflex’s patent was merely the obvious combination of two pre-existing elements and, thus, the patent, upon which Teleflex’s infringement claim was based, was invalid.Patent law relies on the concept of obviousness to distinguish whether new inventions are worthy of being protected by a patent. If a new invention is too obvious, it is not granted a patent and is therefore not a legally protected property interest. However, if an invention is deemed not obvious and has met the other patentability requirements, a patent will be granted, thereby conferring exclusive use of the invention to the patent holder. This exclusive right prohibits others from making, using, selling, offering to sell, or importing into the United States the patented invention [6]. Essentially, the definition of obviousness sets the balance between rewarding new inventions with exclusive property rights and respecting old inventions by not treating minor variations of existing patents as new patents. In this manner, the law seeks to provide economic incentives for the creation of new inventions by ensuring that the property right conferred by the patent will be protected against insignificant variations. The importance of where the line for obviousness is drawn and how clearly it is drawn is especially important in the biotechnology industry. Studies have shown that the development of a new pharmaceutical therapy can take up to 14 years with costs exceeding $800 million [7]. Such an enormous investment of time and money would not be practical if it did not predictably result in a legally enforceable property right.The standard for what constitutes a patentable discovery has evolved over the last 150 years. In 1851, the Supreme Court held in Hotchkiss v. Greenwood that a patentable discovery required a level of ingenuity above that possessed by an ordinary person [8]. Lower courts treated the Hotchkiss standard as a subjective standard, whereby courts sought to determine “what constitute[d] an invention” [9] and a “flash of creative genius” [10]. However, the attempts at imposing the Hotchkiss standard proved unworkable, and in 1952, Congress overrode the case law with the Patent Act, “mandat[ing] that patentability be governed by an objective nonobviousness standard.” [11] This new statutory standard moved the courts away from subjective determinations and toward a more workable, objective obviousness standard.While the Patent Act laid the foundation for the current obviousness standard, the Supreme Court in Graham v. John Deere Co. interpreted the statutory language in an attempt to provide greater clarity as to what exactly “obvious” meant [12]. The Supreme Court determined that the objective analysis would require “the scope and content of the prior art ... to be determined; differences between the prior art and the claims at issue ... to be ascertained; and the level of ordinary skill in the pertinent art resolved.” [13] In addition to analysis under this three-part framework, the Supreme Court called for several secondary considerations to be weighed, including “commercial success, long felt but unresolved needs, [and the] failure of others [to solve the problem addressed].” [13]Unsurprisingly, lower courts were unsatisfied with the Supreme Court’s attempts to clarify the obviousness standard and sought to provide “more uniformity and consistency” to their evaluation of obviousness than the Supreme Court’s jumble of factors provided [14]. In search of consistency, the Federal Circuit created the “teaching, suggestion, or motivation” test (TSM test) “under which a patent is only proved obvious if ‘some motivation or suggestion to combine prior art teachings’ can be found in the prior art, the nature of the problem, or the knowledge of a person having ordinary skill in the art.” [14] Through implementation of the TSM test, the Federal Circuit sought to maintain the flexibility envisioned by the Supreme Court in Graham, while at the same time providing more certainty and predictability to obviousness determinations.The issue before the Supreme Court in KSR Int’l Co. v. Teleflex, Inc. was whether the Federal Circuit’s elaboration on the statutory language of the Patent Act, the TSM test, was consistent with the terms of the Patent Act itself and the Supreme Court’s own analysis in Graham. The Supreme Court determined that while the TSM test was, on its terms, consistent with the framework set out in Graham, the rigid manner in which the Federal Circuit had taken to applying that standard was inconsistent with the flexible approach established by Graham [15]. More generally, it appears the Supreme Court was mainly interested in restoring a more rounded, thorough inquiry to the evaluation of obviousness: “Graham set forth a broad inquiry and invited courts, where appropriate, to look at any secondary considerations that would prove instructive.” [16] As stated by the Supreme Court, “[r]igid preventative rules that deny factfinders recourse to common sense, however, are neither necessary under our case law nor consistent with it.” [17] As such, the Supreme Court reversed the findings of the Federal Circuit, which had found the Teleflex patent valid, and remanded the case back to the lower court with directions to analyze, without rigid adherence to the TSM test, whether the Teleflex patent was obvious [18].The Supreme Court’s ruling in KSR Int’l Co. v. Teleflex, Inc. that the Federal Circuit apply its TSM test less rigidly may have implications for those seeking biotechnology patents in the future. As discussed above, the large investments necessary to develop a marketable biotechnology product demand that entrepreneurs making those investments be reasonably assured that they can predict any future legal hurdles in patenting their invention and in ultimately protecting their patent. As explained by the Biotechnology Industry Organization in its amicus curiae brief in KSR Int’l Co. v. Teleflex, Inc., “[i]nvestment thus is predicated on an expected return on investment in the form of products or services that are protected by patents whose validity can be fairly determined.” [19] Therefore, the Supreme Court’s insistence that the Federal Circuit no longer rigidly rely on the TSM test could increase uncertainty in the grant of future patents. However, the Supreme Court’s refusal to completely dismiss the TSM test, while in fact endorsing its continued use, albeit on a less rigid basis, has to be viewed as a profound victory for an industry with a significant stake in maintaining the status quo. Moreover, it is unclear how much the Supreme Court’s holding in KSR Int’l Co. v. Teleflex, Inc. will truly change the legal analysis of the lower courts, given the evidence that lower courts already were independently shifting away from rigid adherence to the TSM test before the Supreme Court’s ruling [20].More importantly, several aspects of the Supreme Court’s reasoning in KSR Int’l Co. v. Teleflex, Inc. seem to directly address relevant concerns of the biotechnology market in favorable ways. First, the Supreme Court made clear that though a product is the result of a combination of elements that were “obvious to try,” it is not necessarily “obvious” under the Patent Act. Retaining the possibility that “obvious to try” inventions still may be patentable is extremely important to the biotechnology industry in particular because “many patentable inventions in biotechnology spring from known components and methodologies found in [the] prior art.” [21] Rather than foreclosing all “obvious to try” inventions as being obvious, and therefore not patentable, the Supreme Court instead explained that where there is “a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions,” it is more likely that a person of ordinary skill would find it obvious to pursue “known options.” [22] Thus, the proper inquiry, as stated by the Supreme Court, is “whether the improvement is more than the predictable use of prior art elements according to their established functions.” [23] While this reasoning may prevent some “obvious to try” inventions from being patented, it is unlikely to have a substantial effect on inventions in the biotechnology market because “most advances in biotechnology are only won through great effort and expense, and with only a low probability of success in achieving the claimed invention at the outset.” [24] In other words, it would be hard to characterize the use of prior art in the biotechnology context as predictable based on the inherent unpredictability of obtaining favorable results. As such, most biotechnology inventions would presumably fall outside the Supreme Court’s “obvious to try” reasoning due to the very nature of the industry, meaning they would remain patentable under the Supreme Court’s KSR Int’l Co. v. Teleflex, Inc. decision.Second, the Supreme Court recognized the “distortion caused by hindsight bias” and the importance of avoiding “arguments reliant upon ex post reasoning,” though it lessened the Federal Circuit’s rigid protection against hindsight bias [24]. Hindsight bias requires that obviousness be viewed at the time the invention was made, because what may seem revolutionary at the time of invention may, upon the passage of time, seem “obvious.” Cognizance of hindsight bias is crucial for biotechnology patents because “there often is a long ‘passage of time between patent application filing and litigation with biotechnology inventions [that] can exacerbate the problem’ of hindsight bias.” [25] The problem is further exacerbated by the “significantly longer durations of commercial utility” biotechnology inventions enjoy as compared to those in other fields [25]. The more time between the filing of a patent and the subsequent litigation over its validity, the greater the risk that “reliable accounts of [the] context” in which the discovery is made will no longer exist [26]. As such, inventions that were not obvious when they were created will be inescapably colored by the passage of time and by new knowledge and discoveries; the likelihood of this occurrence is higher the further removed the litigation is from the patent filing date. Once again, however, it seems clear that despite the Supreme Court’s abandonment of the TSM test’s rigidity, strong protections against hindsight bias still were emphasized in the Supreme Court’s KSR Int’l Co. v. Teleflex, Inc. decision. In fact, lower courts applying KSR Int’l Co. v. Teleflex, Inc. acknowledge they are “cautious” to avoid “using hindsight” in biotechnology obviousness determinations [27].Finally, the Supreme Court seems to believe that the imposition of a more flexible approach will be more likely to benefit markets not directly at issue in KSR Int’l Co. v. Teleflex, Inc. The Supreme Court asserted, “[t]he diversity of inventive pursuits and of modern technology counsels against limiting the analysis” to the rigid TSM test of the Federal Circuit [28]. This language suggests that the Supreme Court expects lower courts to take into consideration the special considerations facing unique markets, such as the biotechnology market. As such, the specific concerns of the biotechnology market discussed above may receive more attention under the flexible framework asserted by the Supreme Court in KSR Int’l Co. v. Teleflex, Inc.Leading up to the oral argument in KSR Int’l Co. v. Teleflex, Inc., there was widespread speculation that the case could result in a watershed moment, significantly altering the definition of obviousness in patent law. For many, including those in the biotechnology industry, there was ample reason to be concerned. Any change in the definition of obviousness would effectively shift property rights from new patent holders to old, or vice versa. However, the Supreme Court acted with restraint. While the decision purports to make substantial changes by doing away with the Federal Circuit’s TSM test, the opinion seems more like a mild-mannered rebuke of lower courts that had become too complacent in the implementation of their beloved test. If anything, the Supreme Court’s insistence on a more flexible formula is simply a call for lower courts to employ common sense, in addition to considering the factors from Graham and the TSM test. Accordingly, the Supreme Court’s opinion in KSR Int’l Co. v. Teleflex, Inc. is unlikely to have a pronounced effect on the biotechnology market, despite the widespread concern generated before the actual decision was handed down.     

Loss of MT1‐MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid

MT1-MMP (MMP14) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14^−/− mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1-MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1-MMP induces a senescent phenotype characterized by up-regulation of p16^INK4a and p21^CIP1/WAF1, increased activity of senescence-associated β-galactosidase, generation of a senescence-associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14^−/− mice with all-trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions.