Hepatitis E virus (HEV) is one major cause of acute clinical hepatitis among humans throughout the world. In industrialized countries an increasing number of autochthonous HEV infections have been identified over the last years triggered by food borne as well as – to a much lower degree – by human to human transmission via blood transfusion. Pigs have been recognised as main reservoir for HEV genotype 3 (HEV-3), and zoonotic transmission to humans through undercooked/raw meat is reported repeatedly. The minimal infectious dose of HEV-3 for pigs is so far unknown.. Faecal viral shedding and seroconversion was detected in animals inoculated with dilutions of up to 10− 7. This correlates with an intravenously (i.v.) administered infectious dose of only 6.5 copies in 2 ml (corresponding to 24 IU HEV RNA/ml). Furthermore the first detectable shedding of HEV RNA in faeces is clearly dose dependent. Unexpectedly one group infected with a 10− 4 dilution exhibited prolonged virus shedding for more than 60 days suggesting a persistent infection.The minimum infectious dose of HEV-3 in a pig infection model was determined by intravenous inoculation of pigs with a dilution series of a liver homogenate of a HEV infected wild boar. Seroconversion, virus replication and shedding were determined by analysis of blood and faeces samples, collected over a maximum period of 91 days. A dose dependent incubation period was observed in faecal shedding of viruses employing a specific and sensitive PCR methodThe results indicate that pigs are highly susceptible to i.v. infection with HEV and that the swine model represents the most sensitive infectivity assay for HEV so far. Considering a minimum infectious dose of 24 IU RNA/ml our findings highlights the potential risk of HEV transmission via blood and blood products.The online version of this article (10.1186/s12917-018-1713-8) contains supplementary material, which is available to authorized users. Hepeviridae family. Novel taxonomic classification consists of the two genera Piscihepevirus and Orthohepevirus encompassing species A-D. All mammalian HEV isolates have been attributed to species Orthohepevirus A [Hepatitis E is caused by Hepatitis E virus (HEV) which is a major cause of acute hepatitis throughout the world with a total number of 44,000 HEV-related deaths in 2015 [evirus A , 4.Genotype 1 (HEV-1) and 2 (HEV-2) are restricted to humans and are the main cause of endemic outbreaks in developing countries in Asia, Africa and Central America , 6. The HEV genotypes 3 (HEV-3) and 4 (HEV-4) dominate in developed countries and are the main source for autochthonous human cases . In EuroThe HEV-3 transmission from animals to humans via the consumption of infected undercooked/raw meat has been documented in numerous cases , 9. FurtIn general HEV causes a wide range of symptoms, from subclinical to acute hepatitis with icterus up to fulminant hepatic failure : The str− 2 up to 10− 9. Corresponding ct-values, copy numbers and IU are summarized in Table − 2 dilution of the experimentally infected liver contained 9.4 × 105 copies (3.4 × 106 IU) in 2 ml respectively.The infection studies were carried out using tenfold dilutions of the inoculum from 10Through-out the whole observation period none of the animals showed a febrile response or any clinical signs consistent with hepatitis. Additionally, no signs typical for viral hepatitis were seen at gross examination after necropsy.− 7. In the first experimental set up covering homogenate dilutions from 10− 2 to 10− 4 over an observation period of 27 days  followed by groups 10− 3 at 15 dpi and 10− 4 at 17 dpi. Due to this delayed onset it was decided to extend the observation period for the second experimental set up for up to 91 days. The second 10− 4 group started with viral shedding also at 17dpi, followed by the group 10− 5 at 27 dpi. Viruses started to be shed within a time frame of six days for all animals within all groups of the first experiment and these two groups in the second experiment. In the remaining groups inoculated with higher HEV dilutions, a more variable time frame of viral shedding was obvious. In group 10− 6, pig T2–24 shed from 21 dpi - 30 dpi. The remaining animals of this group started one by one after 27 dpi, 34 dpi, and 37 dpi. In group 10− 7 pigs shed the virus until end of experiment; pig T2–17 started shedding virus at 37 dpi, while the other animals in this group started shedding at 55 dpi, 58 dpi and 62 dpi.Viral RNA was detected in the faeces of all groups up to a dilution of 103 HEV copies/μl RNA eventually. Only individual animals of the 10− 4 and the 10− 5 group of the second experiment  in their faeces.The majority of animals had between 1.3 and 51.7 HEV copies/μl RNA in the10% faecal suspension initially and reached a plateau of about 10− 2 in which shedding of HEV RNA in faeces ceased in three out of four pigs  after a mean of 7 d (±2.7). One individual (pig T1–11) remained positive till the end of the observation period on day 23. Unfortunately on day 27 (necropsy) no faeces was available from this animal for testing. In the 10− 3 group only one animal (pig T1–08) cleared the virus before necropsy (27 dpi) 23 days dpi. No viral clearance was observed in the 10− 4 group in both experiments even after 91 dpi. For group 10− 5 an at least 18d period of faecal shedding was seen with no clearance till the end of the observation period of 49dpi. In group 10− 6 virus clearance occurred at very diverse intervals including 11d (pig T2–24), 21d (pig T2–23), 28d (pig T2–22) and more than 35d (pig T2–21), and in group 10− 7 the animals shed virus for at least 36d (pig T2–17) or for 7, 4 or 1 days before being euthanized. Neither in the dilution 10− 8 nor in the dilution 10− 9 signs of virus replication or shedding were observed at all over a period of 76dpi.Viral clearance was detected in group 10− 4/2 , 10− 5  and 10− 7 (pig T2–17) after 24 dpi, 34 dpi and 58 dpi respectively.The detection of HEV RNA in serum was only sporadic and with low viral amounts  seroconversion was determined prior to virus detection which can be explained by the oscillation of corresponding OD values around the cut-off at sampling days 27 and 30 followed by a strong increase from day 34 on. HEV antibodies were assessed by the species independent HEV-Ab ELISA detecting total serum antibodies  in blood ) had a C to T exchange at position 2322. The alignment was done with a previously published full genome (KP294371). The wobble sequence translates into a proline/serine exchange. The point mutation in animal T2–28 was silent.The sequence of the hypervariable region of the inoculum  showed no clinical or molecular evidence for a HEV infection and faecal samples were always negative for HEV RNA. However, one liver lobe of pig T1-01c gave a positive RT-qPCR result (shown in Table ).− 2 dilution up to a 10− 9 dilution and was applied into groups of 4 pigs each. Seroconversion and viral shedding was observed up to a 10− 7 dilution which corresponds to a minimal infection dose of 6.5 copies in 2 ml total volume (according to 24 IU HEV RNA) for swine. In contrast to pigs, the infectious dose for humans is reportedly significantly higher: the transfusion of a platelet concentrate with residual plasma containing 7056–8892 IU HEV RNA was infectious [4 IU [F. Rossi, IPFA [10 copies/ml of a HEV-3 isolate [5 IU/ml for a HEV-4 and 7.51 × 105 IU/ml for a HEV-1 isolate [10 IU/ml) and HEV-3 isolates (2.5–9.5 log 10 IU/ml). In each case cynomolgus monkeys could be productively infected.An increasing number of human hepatitis E genotype 3 (HEV-3) infections has been observed in developed countries in the last decade. This is accompanied by a substantial proportion of HEV RNA positive blood products and a growing number of transfusion transmitted HEV infections . The higfectious . In anotus [4 IU . A minimsi, IPFA . Howeversi, IPFA . In monk isolate  as well  isolate . Similar isolate  using di− 7 dilution lies below the limit of detection of both PCR assays but is sufficient to infect pigs by the intravenous route. This is in accordance with a previous pig titration study where infectivity was observed below the RT-PCR detection limit [− 2 dilution of the inoculum induced viral shedding at 9 dpi, followed by virus shedding at 15 dpi (10− 3 dilution), 17 dpi (10− 4 dilution) and 27 dpi (the 10− 5 dilution) in at least one pig per group, on average 30 dpi (10− 6 dilution) and on average 51 days (10− 7 dilution). The subsequent dilutions also induce growing individual differences in onset of virus shedding varying from 6 to 9 days (10− 4 group), 10–18 days (10− 6 group) up to 16 days (10− 7 group). The increasing extension of incubation period raises the question regarding replication site and persistence of the virus. Further dilutions  of the inoculum induced no HEV infection. In contrast, in the aforementioned study none of the animals inoculated with dilutions below 10− 4 were infected [All infected animals remained asymptomatic and showed no signs of illness such as fever, reduced alertness nor weight loss throughout the experiment which is in line with previous studies in domestic pigs , 25, 28.infected  or 7–14 infected . Using hinfected , 29. In infected . Interesinfected –51. Thisinfected , 52 and infected , which minfected . Howeverinfected . Therefo− 4 group. The animal exhibited a HEV positive liver as determined by necropsy at 27dpi. The first onset of viral shedding in this group was on 17dpi from pig T1–06 which leads to a maximum incubation period of 10 days. The reported periods between oral intake and HEV particles shed in faeces can range from 7.2d [A presumably faecal-oral transmission of HEV was observed by one of the horizontal controls (pig T1-01c) within the 10rom 7.2d  up to 22Antibody response and seroconversion were assessed by two ELISAs with different specifications: The AXIOM ELISA was a multispecies test for detecting total serum antibodies against HEV  whereas the PrioCheck ELISA only detected porcine HEV specific IgG antibodies. Specific porcine IgM ELISAs were not available. In general the serum samples were confirmed positive in the AXIOM ELISA three to ten days before the porcine specific IgG ELISA which is consistent with a primary IgM related immune response followed by IgG response. However, no reliable conclusions can be drawn about the specific occurrence of IgM or IgA or of a mixture of both during the primary immune response. In any case IgA antibodies play an important role in the human immune response against HEV , 56.− 2, one individual from 10− 3 and three from the 10− 6 dilution group cleared the infection within 7d , 8d and 9d up to 27d respectively. All remaining animals were subjected to necropsy prior to a possible virus clearance. All animals of the second 10− 4group displayed a prolonged infection where HEV RNA was detectable in faeces for more than 62 days. Necropsy samples of this group harboured high viral load in bile and liver samples. The 10− 5 group exhibited faecal viral shedding for 22d and individual animals from group 10− 6 and 10− 7 group for 35d and 36d, respectively. Again, all animals showed high viral loads in bile and liver at necropsy. In general, HEV is considered to be a transient infection and viral clearance usually occurs within three weeks after first occurrence in faeces [− 4 group encompassing the whole observation period, to exclude a newly or internal acquired PRRSV and PCV2 infection. Again all samples were negative for both viruses. All animals showing prolonged viral shedding belonged to the dilutions 10− 4 or higher, indicating a correlation between inoculated virus load and prolonged viral shedding. The 10–4 g.Virus clearance could be observed in different groups examined: Three animals in group 10n faeces . The lonn faeces . HoweverAnother research study showed extended virus shedding in wild boars for more than 16 weeks . InteresIt may be of interest that in humans various cases of chronic HEV-3 infections were reported in immunosuppressed patients . For allIn conclusion, this study determined an extremely low minimum infectious dose of HEV to elicit a productive infection in pigs. Additionally our work demonstrated a dose dependent incubation time in pig resulting in a delayed onset of virus replication and shedding. Moreover the study emphasises the potential risk of HEV transmission in humans via blood and blood products. Finally further investigations should be undertaken to determine and compare the infectivity through oral transmission.The inoculum used in this study was prepared from a highly HEV RNA positive liver of an experimentally infected wild boar from a previous HEV-3 infection study .− 2 dilution  to 10− 9. The 10− 2 dilution was sterile filtered prior to inoculation and further dilutions. The corresponding inocula were prepared shortly before inoculation and kept on ice until use and administered intravenously (i.v.) to 4 animals/group.20 g of liver tissue was grounded with mortar, pestle and sterile sea sand. The homogenate was then diluted 1:5 with sterile 1x phosphate buffered saline (1xPBS) to obtain a 20% dilution and centrifuged at 4400 g for 15 min at 4 °C. The supernatant was eventually sterile-filtered through 0.22 μm MILLEX-GP Syringe Filter Unit , aliquoted and stored at − 80 °C. The inoculum was titrated with the Logarithmic dilutions in sterile 1xPBS starting from a 10The competent authority of the Federal State of Mecklenburg Western-Pomerania approved all described animal experiments based on European Directive 2010/63/EU and associated national regulation .− 4 group. A blinded experimental and analysis design was not possible as researchers and animal technicians were supposed to carry out husbandry and experimental work in a defined way in order to mitigate (cross-) contamination risks: every day animal manipulations started in the low dose challenged animals, followed by intermediate dose challenged pigs and in high-dose animals eventually. Moreover, as only standardized quantitative data were obtained  and ELISA derived data), blinding was not considered necessary.For this study 38 domestic pigs (Large White breed) from a commercial breeder  were acquired and housed under containment level 3** conditions. Healthy animals of compatible sizes and ages were allocated randomly to groups by animal technicians. Social incompatibilities were taken into account in few instances for animal welfare reasons. As the study was a titration experiment with a clear cut yes/no readout, the number of animals per group was set as described together with a biostatistician. The experimental design was part of the individualized animal experimental license  which was applied for and given after scientific and ethical assessment by an independent advisory board. After entry, the animals were divided into the experimental groups and held in quarantine for 2 weeks. Individuals of each group were housed together whereas each group was held in a separate stable unit. Complete change of clothes was mandatory before entry of each of the rooms. All animals were pretested for HEV and yielded negative PCR findings in faeces and serum as well negative HEV ELISA results. Due to facility limitations the study was divided in two experiments. The groups were randomly formed consisting of 4 animals each as described in Table Vena cava cranialis.After at least two weeks of acclimatisation negative HEV RNA and Anti-HEV-antibody results were reconfirmed by Rt-qPCR and ELISA. Additionally samples were examined by a novel multiplex PCR   the supernatant was sterile filtrated using a sterile 0.22 μm MILLEX-GP Syringe Filter Unit  and stored at − 20 °C. This solution was the starting point for RNA extraction.At the end of the observation period all animals were slaughtered (electro stunning followed by exsanguination). Animals were euthanized by a veterinarian following EU and German animal welfare regulations and carcasses necropsied by a trained veterinary pathologist assisted by a necropsy technician. Necropsies were performed and samples from blood, faeces and bile as well as different tissue samples were taken for RNA extraction and stored at − 20 °C. Retrieved tissue samples were immediately immersed in 4% neutral buffered formalin.A sample volume of 140 μl of serum, bile and faecal filtrates were extracted manually with the QIAmp® Viral RNA Mini Kit  following the manufactures instructions and eluated in 50 μl buffer. Manual RNA extraction from tissue samples was performed with the RNEasy® Mini Kits . For this purpose 10 mg of a tissue sample was homogenized in 600 μl RLT buffer using TissueLyser II ® (Qiagen). After centrifugation the supernatant was used for RNA extraction according to manufacturer’s instructions. A heterologous internal control  was adde− 4 HEV standard at 7 copies/μl. Viral loads below the lowest HEV standard were retested by an alternative HEV specific qRT-PCR [To monitor the course of the infection a diagnostic quantitative real-time RT-PCR (RT-qPCR) targeting a fragment of ORF3 was performed. Primer, probes and protocol were used as previously reported , 39. Eac qRT-PCR .To monitor the immunological responses, the serum samples were tested with two commercially available HEV ELISA kits. One was the species independent HEV-Ab ELISA   detectinTo monitor and verify the identity of the inoculated compared to the replicated/excreted virus genome a 348 nucleotide long partial sequence of the hypervariable region (HVR) was recovered from corresponding faecal samples. SYBR Green-RT-qPCR followed by a SYBR Green nested PCR both with melting curve analysis was performed as described . The resAdditional file 1:Amplification curve of HEV specific qRT-PCR. A) Amplification curve of HEV specific qRT-PCR targeting the HEV standard (red line) and the RNA extracts from the inocula (blue line). B) Standard curves were obtained by Ct values plotted against the log of starting quantity. C) Obtained Ct values and determined copy numbers (DOCX 219 kb)Additional file 2:Determination of the conversion factor from copies per μl RNA to IU of the WHO standard (DOCX 16 kb)Additional file 3:Calculation of the Infectivity titer of the Liver Used for Inoculation (DOCX 16 kb)Additional file 4:Results of the RT-qPCR from serum and the porcine IgG HEV-Ab ELISA; The graphs display the individual curves of viral RNA in serum and the detection of Anti-HEV-IgG antibodies in correlation to days after infection (DOCX 462 kb)Additional file 5:https://www.geneious.com (DOCX 244 kb)Alignment of the hypervariable region. Alignment done with Geneious version 10.2 created by Biomatters. Available from


Described is an odyssey beginning with interest in colors of ruthenium(ii) complexes and evolving into photochemical uncaging of potent bioregulatory molecules. Over the past several decades, the photochemistry and photophysics of transition metal compounds has blossomed from a relatively niche topic to a major research theme. Applications arising from the elucidation of the fundamental principles defining this field now range from probing the rates and mechanisms of small molecules with metalloproteins to light activated molecular machines. Offered here is a personal perspective of metal complex photochemistry drawn from this author's long involvement with this field. Several examples are described. Topics include characterizing key excited states and tuning these to modify chemical reactivity and/or photoluminescence properties, as well as using photoreactions as an entry to reactive intermediates relevant to homogeneous catalysts. This is followed by discussions of applying these concepts to developing precursors and precursor–antenna conjugates for the photochemical delivery of small molecule bioregulators to physiological targets. The absorption bands that dominate the visible absorption spectra of these Ru(NH3)5L2+, e.g., 1The interaction of light and matter has long intrigued humans of all ages. Indeed this author's personal interest in the photochemistry of transition metal complexes can be traced to the qualitative observation that certain metal compounds left on the benchtop for several days looked very different than those kept in the dark for a comparable period. This was during a postdoctoral fellowship in Henry Taube's Stanford U. laboratory, where as a new convert to inorganic chemistry, I became fascinated by the bright colors of transition metal complexes. The compounds of interest were the pentaammineruthenium(ii) ammine complexes, once I joined the chemistry faculty of the University of California, Santa Barbara (UCSB). I was fortunate that several UCSB colleagues were studying gas phase photoreactions and, moreover, were willing to teach this novice some nuances of photochemical science as it was then practiced. To set the context, I should emphasize that there had already been significant activity investigating photoreactions of metal complexes. Much of this work has been summarized in books by Balzani and Carassiti (1970)iii) oxalate-based actinometry developed by Hatchard and Parker in the UK (1956).It was with these materials that I first noted light induced color changes and learned to store my samples in the dark. Furthermore, curiosity about the underlying causes led me to begin investigating the photoreactions of the Ru5(py-X)2+ is “what excited states (ES) are involved?” From the orbital parentage of these ES, one might develop a qualitative view of expected reaction trajectories. Given the power of density functional theory (DFT) computations now accessible, such visualization might seem primitive, but it does provide intuition of the type that has made the practice of chemistry a very human activity. Stepping back to our earliest photochemical investigations, neither DFT nor laser flash photolysis were readily available. As a consequence, mechanistic conclusions were largely built on determining photoreaction products and quantum yields and on comparing these data to the electronic spectroscopic properties.The first question one might ask when considering the solution photochemistry of a complex ion like Ru5(py-X)2+ and related complexes such as Ru(NH3)5(pz)2+ (pz = pyrazine), the strong absorption bands (ε ∼ 104 M–1 cm–1) seen in the visible spectra were assigned as spin-allowed (singlet to singlet) MLCT transitions, while those in the ultraviolet region (UV) as IL (πL → π*L) in character. Where would one expect to find the LF absorptions for these low spin d6 complexes? As an empirical model, one might consider the spectrum of Ru(NH3)62+, which does not include any π-unsaturated ligands. The electronic spectrum of this octahedral complex displays two relatively weak absorption bands at 390 nm (ε = 35 M–1 cm–1) and 275 nm (640 M–1 cm–1) as predicted by group theory (1A1g → 1T1g and 1A1g → 1T2g)3)5(py-X)2+ ions, but they are obviously obscured by the much stronger MLCT bands.For Ru(NHi as shown in eqn (1). Note that Φi is unit-less. An efficient process has a quantum yield near 1 (or higher if a chain reaction results); however, as discussed below, there are many reasons why ΦA might be much smaller. A key point here is the nature of the denominator, since photoreactions will not occur unless light is absorbed. Quantum yields are typically measured at a specific wavelength of irradiation (λirr), so this concept is less precise when a broadband excitation source such as sunlight is used.The quantum yield is basically an efficiency measurement and can be defined for a specific photoprocess rate of the photochemical reaction is of key interest. The photoreaction rate equals Φi × Ia, where Ia is the intensity of the light absorbed, typically in units of einsteins per unit time (1 einstein = 1 mole of photons) or in einsteins per unit volume per unit time. For a solution where the photoreactant is the only species absorbing the incident light, Ia = I0(1 – 10λ)–Abs, where Abs(λ) is the solution absorbance at λirr and equals the product of the molar concentration of the photoactive species (c), the molar extinction coefficient  at that λ and the path-length of the cell (in cm). There is no unimolecular rate constant associated with this process.With certain applications, for example, the uncaging of a drug at a physiological target, the overall 3)5(py)2+, continuous photolysis of this ion with 405 nm light in ambient temperature, aqueous solution resulted in the photosubstitution reactions depicted in eqn (2).Φpy and Φ3NH were measured as 0.045 and 0.063 for λirr = 405 nm. This excitation wavelength is close to the λmax of the spin-allowed MLCT band (407 nm), so one can be assured that the vast majority of the photons absorbed lead to MLCT excitation.Taking for example Ru(NHIII(py–)]2+? Given that low spin d5 Ru(iii) complexes are not very thermally labile, this behavior is not what one might expect for the MLCT* state. In contrast, the MC* states, which involve promoting an electron from a non-bonding or even π-bonding orbital to one which is σ* relative to the metal–ligand bonds would appear to be logical precursors of ligand photosubstitutions. Consistent with this argument, the hexaammine complex Ru(NH3)62+ is comparably labile toward photodissociation of NH3 (Φ3NH = 0.27 for λirr = 405 nm).3MC* state of Ru(NH3)5(py)2+ confirms that this state is nearly dissociative in character.9Are the observed photosubstitution reactions consistent with that expected for an MLCT excited state, which can simplistically be represented as having an oxidized metal center and a reduced pyridine,  using Stern–Volmer plots of τ0/τ vs. [Q] according to eqn (9) where τ0 is the lifetime in the absence of quencher. The value of kq will depend on the viscosity of the solvent (hence the rate of diffusion) and the operating quenching mechanism(s). If the latter involves energy transfer, the rate will depend on the spectral overlap between the donor and acceptor pair as well as the energies of the relevant excited states. If it involves electron transfer, then the key issue will be the relative redox potentials of the excited state species and of the quencher. We illustrate these points with photo-luminescent Cu(i) complexes.From a kinetics perspective, quenching may have a major impact on the luminescent lifetime 10 metal systems offer an opportunity to observe other excited states.i) complexes of the type Cu(DMP)2+ ion  described by McMillin et al.i) complexes, tetranuclear clusters such as Cu4I4py4 that consist of Cu4 tetrahedra with short Cu–Cu distances. Bridging iodides and terminal pyridine ligands complete the unit. Related polynuclear copper(i) clusters have drawn recent interest for potential applications in OLEDs.41Luminescence in first row transition metal complexes is frequently bypassed by the presence of lowest energy MC states that are too short-lived to have measurable emissions. Thus, filled shell d4I4py4 demonstrate marked environmental sensitivity, i.e., the luminescence spectra change reversibly by varying the temperature and the rigidity of the medium4I4py4 displays two distinct emission bands that also demonstrate different lifetimes (3)63+ and Ru(bpy)32+ ions described above, where single emissions from lowest energy MC and MLCT states were respectively recorded.The photophysical properties of Cuifetimes . This beτ = 10.6 μs) lower energy (LE) emission (λmax = 690 nm) from Cu4I4py4 in 296 K toluene solution was assigned to a triplet cluster-centered (3CC) ES, resulting from a combination of delocalized iodide-to-copper charge transfer and d–s transitions.τ = 0.45 μs) higher energy (HE) band (λmax = 460 nm) was assigned to a triplet halide-to-ligand (pyridine) charge transfer (3XLCT) ES.The strong, longer-lived  to orbitals that are bonding with respect to the Cu4 cluster. As a result the surfaces of the 3CC and 3XLCT states overlap poorly, and there is an energy barrier for internal conversion from the 3XLCT state to the lower energy 3CC excited state )chromium(iii) derivatives CrL3.3 complexes quench by competitive energy transfer and electron transfer mechanisms  of the lowest ES of this species, a Cr(iii) centered 2MC state, is essentially unchanged.46The kinetics of biomolecular quenching of the Cuchanisms . By vary3CC state is ∼1.66 μm–1 (2.06 V) and the excited state reduction potential E1/2([Cu4I4py4]+/[Cu4I4py4]*) is ∼–1.78 V (vs. the ferricenium/ferrocene couple). Thus the 3CC state is a very powerful reducing agent, but it also is capable of undergoing energy transfer quenching by the CrL3 centers. The bimolecular rate constant for the latter process was shown to be ∼107.9 M–1 s–1, while contributions to the quenching by an electron-transfer mechanism were evident for those Q with reduction potentials E1/2(Q/Q–) less than 1.4 V. This behavior is demonstrated by the plot of log10(kq) vs. the ΔGo of the electron transfer quenching step calculated according to eqn (10)  . The curvia internal conversion to lower energy states of the same spin multiplicity and/or intersystem crossing to lower energy ES of different multiplicity(ies). From either type of ES, luminescence , non-radiative deactivation to the GS and/or chemical reaction can occur. The focus here will be on the reactive pathway, since primary photoproduct(s) formed, while no longer electronically excited, may yet be quite reactive under the experimental conditions. Thus, the final products observed may result from secondary pathways of the intermediate(s) I.Within the anatomy of a photoreaction, there are potentially a number of steps between the initial ground state and the final product(s) as qualitatively illustrated in I, which then can be characterized by conventional spectroscopy. An alternative (and complementary) approach is to use flash photolysis to generate such species and fast detection techniques to characterize their spectroscopy and reaction dynamics as they evolve to the final products.There are several ways to probe such intermediates. For example, in low temperature matrices, the activation energies for surmounting the barriers illustrated in Our first flash photolysis apparatus was a “conventional” flash lamp system with a design not altogether that different from that first built by Norrish and Porter, who were awarded the 1966 Nobel Prize in Chemistry for their contributions to fast reaction kinetics. This system operated by discharging hundreds of Joules of electrical energy from a capacitor bank through 10–20 cm long cylindrical quartz flashlamps filled with xenon gas. The white light flash lasted about 10 μs and irradiated a sample cell with a path of similar length. If one were sufficiently careful to avoid saturating the PMT detector with scattered light, it was possible to record flash-induced temporal changes in the transient spectra.3)5(py)2+ ion that had first stirred our interest in photochemistry. Our early flash studies indicated the formation of a transient species that partially returned to the starting material on a millisecond time scale.via formation of a transient species by which the Ru–py bonding has isomerized from monodentate coordination to a π-complex such as the η2-py species illustrated in ii) coordination at a pyridine C–C bond was concluded to be the most stable η2-pyridine complex.The first system that we used this flash photolysis apparatus to study was the highly colored Ru species Rh(PPh3)3Cl, first reported by Wilkinson and coworkers.3)2Cl formed by spontaneous dissociation of PPh3 (trans-Rh(PPh3)2(CO)Cl (kCO = 7.8 × 108 M–1 s–1). By carrying out the experiment in the presence of reactants such as PPh3, H2 and ethylene, it was possible to determine other second order rate constants for trapping the Rh(PPh3)2Cl intermediate.One such study was concerned with the reactivity of intermediates proposed in the homogeneous hydrogenation of olefins by the rhodium( of PPh3 . One way)2(CO)Cl . We studkL for the reaction with PPh3 was determined to be ∼6 × 107 M–1 s–1 in 296 K benzene while that with H2 was nearly a factor of 30 slower. In addition, kCO values in cyclohexane and dichloromethane were ∼2-fold and 5-fold lower, respectively, suggesting that the intermediate is not three-coordinate, but is weakly bound by solvent in the position vacated by CO.53For example, the νCO region of the IR spectrum.While flash induced changes in the optical spectra are often particularly valuable for following the dynamics of excited states and reactive intermediates, the interpretable structural information from the transient spectra is often limited by the broadening of the absorption and emission bands in solution. In this regard, other time-resolved spectroscopic techniques can provide new dimensions. Time-resolved infrared (TRIR) spectroscopy is especially effective in probing fast reactions of ES and intermediates generated by the photo excitation of metal carbonyls.νCO region upon 308 nm flash photolysis of CH3Mn(CO)5 in cyclohexane solution.cis-CH3Mn(CO)4(Sol) (eqn (11), Sol being the solvent). Homolytic fragmentation of the Mn–CH3 bond was a minor (<10%) photochemical pathway as well.An example is shown in 3Mn(CO)5 at rates linearly dependent on [CO] and strongly affected by the nature of the solvent. In cyclohexane, the second order rate constant kCO (295 K) was 4.5 × 108 M–1 s–1, while the value in THF (1.4 × 102 M–1 s–1) was more than 6 orders of magnitude smaller. For reactions in THF, the temperature dependence of kCO was investigated over the range 232–293 K, and an Eyring plot gave ΔH* = 76 kJ mol–l and ΔS* = 59 J mol–l K–l.S* value suggests that considerable Mn–Sol bond breaking is occurring in the rate-limiting step of this substitution pathway.As other studies have shown, the empty site generated by CO photodissociation is often filled by coordination to a solvent molecule, even ones as weakly binding as a saturated hydrocarbon.3Mn(CO)5 were extensively probed as mechanistic models for this important class of organometallic reactions.cis carbonyl leads to a reactive intermediate in a step promoted by more polar solvents.Another reaction of interest from the catalysis perspective is the “migratory insertion” of carbon monoxide into metal alkyl bonds (eqn (12)). This is the key carbon–carbon bond formation pathway in catalytic carbonylations such as acetic acid synthesis from methanol and alkene hydroformylation.I (eqn (13)) with the same composition as the intermediate proposed for migratory insertion. Time-resolved optical and IR spectral studies were then used to interrogate I and the dynamics of the reactions with various L′ to give the stable acyl products and of reverse alkyl migration to give the metal alkyl complex M–R.Our strategy for characterizing the structure and reactivity of such intermediates started with the acyl complex. Photodissociation of a ligand L′ from the acyl complex M-C(O)R prepares a reactive species I in this manner to the competitive reactivities deduced for intermediates in the thermal migratory insertion kinetics provide guidance relevant to mechanisms of these reactions. We studied such migratory insertion pathways by starting with Mn(CO)5(C(O)R),5-C5H5)Fe(CO)(C(O)R),3C(O)Co(CO)3PPh3 (3Ir(CO)2I3]–.Comparisons of rates measured for CO)3PPh3  and  while formation of CoM was independent of [CO]  in accord with the scenario illustrated in kCO and kM indicate that the η2-acyl is indeed the most likely structure for CoI.68CoI. The solvent insensitive kCO has a very small ΔH‡ (6 kJ mol–1) but a sizable, negative ΔS‡ (–92 J mol–1 K–1), and these suggest an associative mechanism to regenerate CoA. The kM rates to give CoM are accelerated by donor solvents and reflect more intimate involvement of solvent in the methyl migration. This may be necessary owing to the orientation of the alkyl group in CoI that is not well suited for migration to the metal center.Regeneration of Nitric oxide plays major roles in cardiovascular regulation, and one key bioregulatory target is the ferroheme protein soluble guanylyl cyclase (sGC), which displays a remarkable specificity for NO.iii) complex Ru(NH3)63+ in acidic aqueous solution (eqn (14)). They measured a rate constant (kNO = 0.2 M–1 s–1 at 298 K), which was much larger than that for NH3 substitution by other ligands. They concluded that the reaction with NO must be associative, whereby the low spin d5 Ru(iii) center engages the odd electron of NO to give a 7-coordinate Ru(NH3)6(NO)3+ transient. Subsequent activation parameter measurements gave a relatively small ΔH‡ (41 kJ mol–1) and a large and negative ΔS‡ (–114 J K–1 mol–1)V‡ (–14 cm3 mol–1)One precedent is the report by Taube and coworkers5 Fe(iii) porphyrinato complexes? To probe this question, we investigated the flash photolysis kinetics of aqueous solutions containing FeIII(TPPS) (TPPS = tetra(4-sulfonato-phenyl)porphyrinato) and NO, which are in labile thermal equilibrium with k11 = 103 M–1 (298 K).III(TPPS)(H2O)(NO), followed by a relaxation of the system back to equilibrium (eqn (15)). Under excess NO, the latter process is exponential. Linear plots of the resulting kobs values vs. [NO] follow the relationship kobs = koff + kon[NO] from which the values of koff = 0.5 × 103 s–1 and kon = 4.5 × 105 M–1 s–1 (298 K) were determined.The temperature dependence of these rate constants over the range 298–318 K, gave the ΔH‡ and ΔS‡ values for the NO “on” and “off” pathways of FeIII(TPPS) as 69 kJ mol–1 and +95 J mol–1 K–1 for kon and 76 kJ mol–1 and 60 J mol–1 K–1 for koff, respectively.83Should a similar mechanism be valid for the high spin dV‡, which reflects the sensitivity of the rate constants to pressure (ΔV‡ = –RT(d(ln k))/dPhyd)T, where Phyd is the applied hydrostatic pressure. Pressure effects on laser flash photolysis kinetics were measured in these laboratories using the laser flash photolysis cell illustrated in kobs over a range of [NO] to determine kon and koff for each hydrostatic pressure investigated. Plots of ln(kon) and of ln(koff) vs. P were linear in this pressure region and from these ΔV‡on = +9 cm3 mol–1 and ΔV‡off = +18 cm3 mol–1 were determined.83Another parameter is the activation volume ΔIII(TPPS)(H2O)2 will exchange its coordinated waters with solvent much faster than the reaction with NO, which was present at mM concentrations. This indeed is the case; H2O exchange with FeIII(TPPS)(H2O)2 occurs with a reported first order rate constant (kex = 1.4 × 107 s–1 in 298 K water)kobs values determined at any [NO].These activation parameters suggest the reaction pathways envisioned in eqn (16) and (17). This pathway also predicts that FeH‡ex = 67 kJ mol–1, ΔS‡ex = +99 J mol–1 K–1 and ΔV‡ex = +7.9 cm3 mol–1, which are in remarkable agreement with the activation parameters measured by flash photolysis for the kon pathway with NO.III(TPPS)(H2O)2 therefore dominate solvent exchange in agreement with a dissociative mechanism, the limiting step being eqn (16). Not surprisingly, the substitution mechanism for the labile, high spin d5 FeIII(TPPS)(H2O)2 complex is markedly different from the associative mechanism described above for the low spin d5 Ru(NH3)63+ ion.A more recent reexamination of the exchange reactionII(TPPS) gave the much larger value of kon = 1.5 × 109 M–1 s–1 (298 K), and a value of koff so small (6.4 × 10–4 s–1) that it had to be measured by a different technique.kon value for the ferrous complex is much less temperature sensitive (ΔH‡on = 24 kJ mol–1) consistent with a reaction pathway having a rate approaching the diffusion limit in aqueous solution. Analogously large kon values have also been reported for the ferrous heme protein sGC.91Studies with met-myoglobin came to similar mechanistic conclusions for the reactions of this ferri-heme protein with NO.In many fundamental investigations, there is often later recognition that the knowledge gained can be utilized for practical applications not perceived at the onset. This is especially true in chemistry, a discipline that lies at the interface between truly fundamental science and the chemical, materials and pharmaceutical industries. Indeed, many practitioners of transition metal photochemistry have for some time been interested in developing methods for converting solar radiation to chemical potential energy, for designing new photo-optical materials and for improving the efficiency of lighting with new types of OLEDs. Biomedical applications include photodynamic therapy, new photoactivated anti-cancer drugs and the photochemical delivery (photo-uncaging) of bioactive substances to physiological targets.location and timing, but also of the dosage for SMB delivery. Thus, photo-uncaging has value both as an investigative tool in physiology and for its ability to affect the progression of specific disease states.Our focus in this regard has been on the photo-uncaging of the small molecule bioregulators (SMBs) nitric oxide and carbon monoxide. This technique has the potential to provide unprecedented control, not only of the NO, formed endogenously by several isoforms of the enzyme nitric oxide synthase (NOS), has numerous roles in mammalian biology including cardiovascular regulation and immune response. Among potential therapeutic applications of controlled NO delivery are cardiovascular treatment, antibacterial control of biofilms and toxic effects on cancer cells.e.g., eqn (15)).2[Fe2S2(NO)4], also know as Roussin's red salt (RRS).iii) nitrite complexes.Two themes have guided our photochemical studies of metal nitrosyls. One, described above, used flash photolysis to investigate the mechanisms of ferri- and ferro-heme reactions with NO  spectral region (∼700–1100 nm).106There are several approaches to the problem of limited tissue transmission at certain wavelengths. For example, one might design a small polymeric device attached to an implantable optical fiber.e.g. aerated, aqueous media at 37 °C) and (ii) the absence of undesirable toxicity of the photochemical precursor or of the residual photoproduct once the SMB is released. Also desirable are (iii) the ability to activate SMB release using longer wavelength, esp. NIR, excitation, and (iv) to track the location of these species in the organism and to determine whether the system has indeed undergone photo-activated release. In this regard, photoluminescence (PL) is a particularly sensitive imaging method. Lastly, (v) spatio-temporal control of SMB release is essential, and while this is partially achieved by using light as the trigger, developing delivery mechanisms that can target specific sites will enhance efficiency and reduce undesirable side effects.In order to simplify terminology we will use “photoCORM”, a term we coined several years ago,Following the earlier studies referenced above, we and others developed a number of transition metal containing complexes and materials that release NO when activated by light. These studies have been summarized in various reviews,iii) O-nitrito complex trans-Cr(cyclam)(ONO)2+  meets several of the desired criteria. It is photoactive with quantum yields measured by spectral changes of 0.25–0.30 moles per einstein over the λirr range 365–536 nm and NO is a photoproduct.ΦNO = 0.25 when the photoreaction was carried out in the presence of added glutathione (GSH). Photolysis of low concentration CrONO solutions released sufficient NO to activate the key enzyme soluble-guanylate cyclase (s-GC) and, in separate experiments, to contract relaxed endothelium-free porcine arteries with an IC50 concentration ∼320 nM.110The chromium(iii) nitrito complex might undergo CrO–NO photofragmentation to give NO plus a CrIV(O) species.3)5(ONO)2+, while showing some NO release, primarily led to ligand photolabilization.trans-Cr(cyclam)Cl2+ is relatively inert to photosubstitution, although other chlorido complexes such as Cr(NH3)5Cl2+ are not.iii) MC excited states requiring substantial distortion along reaction channels involving the equatorial ligands.2MC state of the Cr(iii) center.110The discovery of CrONO's photochemistry was not accidental. There were previous indications that a Cr to regenerate CrONO. Permanent photochemistry is only observed when the NO is removed by entraining the solution with air or helium, or if a trapping agent intercepts the CrIV(O), thereby preventing the back reaction. O2 serves this purpose. Thus photolysis of CrONO in aerated aq. solution leads to permanent photochemistry, presumably via oxidation of Cr(iv) to Cr(v). However, the net NO production is inefficient owing to scavenging by the superoxide presumably also generated in this step. On the other hand, when mM concentrations of the common cellular reductant GSH are present, reduction back to a Cr(iii) product dominates (110The Crominates .110ε); they are Laporte-forbidden transitions for this centro-symmetric complex. Thus, the rate of NO generation is relatively slow unless the CrONO concentration and/or excitation light intensity is high. To address this issue, we decided to attach various organic chromophores to the CrONO-type platform to serve as antennas to enhance light absorption and, correspondingly, NO production  as photosensitizers, since QD absorption and emission energies can be tuned through the visible spectrum by varying the nanoparticle diameters.2(μ-RS)2(NO)4), prepared from the RRS anion by the reaction shown in eqn (19). These RSE are more stable than is RRS and upon photolysis in aerated solution release all 4 NO (eqn (20)). Variations of this synthesis were used to build RSE's with strongly absorbing chromophores, an example being PPIX-RSE (rate of NO release from PPIX-RSE versus that from a simpler RSE such as Fe2(μ-SEt)2(NO)4 (Et-RSE) is largely due to more efficient light absorption (Ia) by the tethered antenna. Energy transfer from PPIX* to the cluster is also evidenced by the quenching of the characteristic PPIX PL (4S3(NO)7] in a nanocarrier made from carboxymethyl chitosan; however, the excitation wavelength was the near UV.In parallel studies, we investigated the antenna effect with Roussin's red salt esters  bipyridyl complexes. In this laboratory, we are investigating two multi-photon approaches: simultaneous two-photon excitation (TPE) and energy transfer upconversion (ETU), which functions by sequential photon absorptions. Unlike the single photon excitation (SPE) we have been discussing, the rates of TPE and ETU processes have a non-linear dependence on the incident light intensity I0. For example, the probability of TPE is proportional to I02, if a single source is utilized. Thus, TPE induced photoreaction occurs mostly at the focal point of the excitation source, offering three-dimensional spatial resolution, a property widely exploited in imaging. Such resolution would provide unprecedented definition of the uncaging location.123None of the photoNORMs described above displayed significant absorbance at the NIR wavelengths where tissue transmission is optimized. In principle, this limitation can be addressed by using multi-photon excitation, where the summed energy of several NIR quanta is sufficient to populate the higher energy excited states from which the desired uncaging occurs. See, δ) as large as 104 GM  seen for CdSe nanoparticles.125The selection rules for TPE are different from those for SPE. The former is allowed only between two states that have the same parity; the latter requires a parity change.λmax 632 nm) when subjected to 810 nm excitation with 100 fs pulses from a Ti/sapphire laser.δ value for PPIX is quite low (∼2 GM).Notably, aqueous solutions of PPIX-RSE that show no absorbance at NIR wavelengths, display a weak PL 2 dependence predicted for a photochemical process initiated by TPE.Fluorescein has a higher Subsequent studies have confirmed TPE with pulsed NIR lasers for other RSE derivatives with chromophores having large two photon cross-sections.129I0 values are needed. Instead of pulsed lasers, such NIR to visible/UV upconversion can be achieved with relatively inexpensive continuous wave (CW) NIR diode lasers. If the desired upconversion involves the net of two photons, the intensity dependence can vary from second order at low I0 to first order at very high I0 . This depends upon the rates of the competing energy transfer and deactivation pathways. The typical situation is between these limits, and as a consequence, the potential for 3-D resolution using light source focusing remains.Multi-photon NIR excitation of caged NO and other bioactive molecules can also be achieved by energy transfer upconversion. The advantage of ETU is that it involves sequential, rather than simultaneous excitation, so lower III ion such as Yb3+ is the sensitizer that absorbs the NIR light. This energy is transferred to an activator such as Er3+ as shown in 3+ and energy transfer to the long-lived excited state of the activator populates higher energy states from which visible/UV emission occurs. If an appropriate solid matrix is used and the core is coated with a shell of host lattice, silica or polymer, deactivation pathways are suppressed and stronger upconverted emissions result.In order to exploit ETU, we collaborated with Fan Zhang and Dongyuan Zhao from Fudan U. (China) and UCSB colleague Galen Stucky to test lanthanide ion-doped upconverting nanoparticles (UCNPs) as NIR antennas for NO photo-uncaging. With such UCNPs, a Lnet al.4 : Yb/Tm UCNPs to uncage acetate from 3′,5′-(carboxymethoxy)benzoin acetate loaded on the surface, and in 2012 Y. Yang et al.d-luciferin in C6 glioma cells and in living mice. Notably, the latter studies showed that NaYF4 (Yb3+/Er3+) UCNPs are non-toxic, their components are eventually excreted, and their location in the organism can be imaged by PL.There was limited precedent for using such UCNPs for uncaging bioactive substances with NIR excitation. In 2010, Carling 4 : Yb/Er : NaYF4 core : shell UCNPs triggers NO release from the anion of Roussin's black salt Na[Fe4S3(NO)7] (RBS). Solutions of RBS are photoactive under visible excitation (eqn (21))3+ groups to render the nanoparticle water-soluble and to facilitate ion pairing with the RBS anions. As shown in 4 : Yb/Er UCNPs, so the emitted light can be reabsorbed by RBS to facilitate the photoreaction. The NO output as measured with the NOA was linear with the irradiation time at constant power but non-linear in response to systematic increases in excitation intensity (∼I01.5).Our first UCNP uncaging study, also reported in 2012,A more robust nanocarrier is illustrated in et al.The mechanism by which UCNPs sensitize NIR mediated NO release from photoNORMs has not yet been well defined. Although there have been reports of Förster resonance energy transfer involving UCNPs,4 : Yb/Er UCNPs.Although the UCNP emission/photoNORM absorption may not be the most efficient, this pathway simply requires a precursor having strong absorption bands that overlap with the UCNP emissions. This concept led our laboratory to develop biocompatible polymer-based carriers in which the UCNP and the photoNORM are simply co-dissolved. The first examples are mm-sized polymer disks consisting of polydimethylsiloxane (PDMS) containing RBS and NaYF2–. More direct NO detection is now available using copper(ii) complexes with chelating ligands that have pendant luminophores.ii) largely quenches the emission from these species owing to its low energy LF transitions, but reaction with NO both modifies the ligand to enhance its PL and reduces the metal center to Cu(i), thereby deleting these low lying transitions.142Another key issue in therapeutic NO delivery is detection and analysis. The Sievers NOA mentioned above measures NO that has been entrained into the gas phase from a solution or an organism. Although very accurate and sensitive, this method is transport-limited and not very useful for real time studies. There are also sensitive NO specific electrodes that are effective in solution media, although in our hands there were issues with reproducibility.in vitro test for CO is the carboxymyoglobin (Mb-CO) assay, where CO is added to a solution of reduced deoxy-Mb and formation of Mb-CO is followed via changes in the absorption spectrum.Although our investment in developing photoCORMs has been less than with NO releasing analogs, the design principles carry over as do some problems, among them the need for reliable detection methods.ΦCO) in some cases exceeded 1.0. The high yields of CO release can be attributed to subsequent reactions of initial photoproducts with O2 and/or the aqueous solvent  complex Re(L)(CO)3(bpy)+ (L = P(CH2OH)3).τ = 400 ns) in solution and releases one CO upon photolysis (eqn (22), ΦCO = 0.11). The photoproduct Re(L)(CO)2(H2O)(bpy)+ is also luminescent. Neither Re(L)(CO)3(bpy)+ nor its photoproduct displayed any acute cellular toxicity up to 100 μM. Thus, we were able to use confocal microscopy to observe the PL of the rhenium species incorporated into cells (human prostatic carcinoma cell line PPC-1) before and after photolysis (A very interesting photoCORM developed at UCSB is the rhenium(otolysis . The abi3(bpy)+ can be attributed to metal-to-bipyridine charge transfer states. However, CO photolability occurs primarily from metal-centered ligand field states.i) complexes to the red, but excitation at those longer wavelength bands does not lead to significant CO release, since this does not populate the higher-energy MC states necessary for CO labilization. In fact, the story is quite analogous to the photoreactivity of the Ru(NH3)5(L)2+ complexes discussed above.10The task remains therefore to develop a mechanism to release CO with NIR light. In this context, the photophysics of metal carbonyls offers a challenge. The stronger lower energy absorptions and the PL of diimine complexes such as Re(L)(CO)i) complex similar to the Re(i) complex just described. Manganese, however, has smaller d-orbital splitting than the heavier transition element analogs. Thus, it has inherently lower energy LF states, thereby making CO photodissociation energetically more accessible.We have addressed these issues by designing a nanocarrier that incorporates both a photoCORM and a UCNP antenna to mediate multi-photon NIR labilization of CO. The photoCORM is a Mn(i) photoCORM trans-Mn(CO)2(PPh3)2(bpy)+ displays strong visible range absorptions, photolysis of which leads to facile CO release. This spectrum is well suited for matching the upconverted emissions from a NaYF4(Yb20Tm0.2) UCNP. In collaboration with Nanfeng Zheng of Xiamen U (China), we assembled nano-carriers consisting of a UCNP core coated with a phospholipid-functionalized with poly-(ethylene glycol) (DSPE-PEG 2000) (trans-[Mn(CO)2(PPh3)2(bpy)](CF3SO3) is readily infused. NIR excitation (980 nm) of an aq. solution containing the loaded nano-carrier leads to CO release. With these hydrophobic components, leaching is minimized, while encapsulation isolates them from the medium, thereby reducing potential toxicity and promoting higher loading. This remarkable nano-carrier brings together the UCNP NIR antenna and a hydrophobic photoCORM in a water-soluble ensemble.For example, the Mn(EG 2000) .152 The We are extending these studies to examine the efficacies of such nano-carriers with other photoCORMS and photoNORMs and with UCNPs responsive to 800 nm excitation. In addition, surface modifications should be well suited for the control of targeting and other properties of these units.in vitro and in vivo.et al., used micelles constructed of peptide terminated polymers for the targeted delivery of chemotherapeutic agents to malignant gliomas, thereby minimizing other side effects.154While photo-excitation allows one to define location and timing of SMB uncaging in tissue, a key remaining task is to improve transport of the precursor–antenna conjugates to the desired targets. Although one can envision implanting a device such as the mm-sized PDMS mini-disks described above, a more elegant strategy would be to employ biological mechanisms for this purpose. We are embarking on this quest. One approach is to use targeting peptides that, depending on the amino acid sequence, can direct water-soluble nano-carriers to specific cell types both In collaboration with UCSB colleague Norbert Reich, we have constructed nano-carriers with hollow gold nanoparticle (HGN) cores (∼40 nm diameters).140Based on this precedent, our premise is that decorating photoCORM and photoNORM nano-carriers with a targeting polypeptide will greatly enhance delivery specificity. The novelty here lies not in using these targeting polypeptides, but in combining this methodology with the uncaging of SMBs from NIR responsive conjugates with UCNP cores. The residual PL from UCNPs offers imaging opportunities as well. Such devices will provide imageable and unprecedented spatio-temporal control of SMB delivery to specific cell types.This article illustrates the evolution of research into the photochemistry of transition metal chemistry by tracing the author's interest and involvement in this area over four decades. Initial studies focused on correlating reaction product characterizations and quantum yields with spectroscopic properties to identify the excited states responsible for specific photochemical processes. This information was then used to tune excited state energies in order to correspondingly direct the resulting photoreactivities. Within the same time frame, related studies were directed toward elucidating specific mechanisms for excited state reactions and deactivation pathways. The availability of flash photolysis methods provided access to the dynamics and trajectories of excited state pathways as well as of subsequent reactions of transient intermediates formed. Notably, such species are often analogous to intermediates proposed for certain thermo-chemical processes, so pulsed photolysis offers an important mechanistic tool in catalysis and bioinorganic chemistry. Our personal journey in this area has turned to using photochemical methodologies for the uncaging of bioactive small molecules, with the eventual goal of establishing guidelines for therapeutic application.Another researcher would undoubtedly provide a different perspective, given that individual's choice of other research problems and tools employed. Regardless, it is clear that the successful use of inorganic photochemistry, whether in energy science, biomedicine, or other foreseen or unforeseen application, needs to be based on a sound understanding of the fundamental principles that define the mechanisms of these systems. I am convinced that, as new tools and ideas are introduced, fascinating and unexpected developments in the photochemistry and photophysics of transition metal compounds will continue to be forthcoming.


Rising [CO2] and temperature will alter the growth and productivity of major food and forage crops across the globe. Although the impact is expected to be greatest in tropical regions, the impact of climate-change has been poorly studied in those regions.By mid-century, global atmospheric carbon dioxide concentration ([CO2] (600 μmol mol−1) and warming (+ 2 °C), singly and in combination, on Panicum maximum Jacq. (Guinea grass) metabolite and transcript profiles.This experiment aimed to understand the effects of elevated [COPanicum maximum transcriptome. Leaf samples were taken at two time points in the Guinea grass growing season to analyze transcriptional and metabolite profiles in plants grown at ambient and elevated [CO2] and temperature, and statistical analyses were used to integrate the data.We created a de novo assembly of the 2] combination plots.Elevated temperature altered the content of amino acids and secondary metabolites. The transcriptome of Guinea grass shows a clear time point separations, with the changes in the elevated temperature and [CO2] result in alterations in transcript and metabolite profiles associated with environmental response, secondary metabolism and stomatal function. These metabolic responses are consistent with greater growth and leaf area production under elevated temperature and [CO2]. These results show that tropical C4 grasslands may have unpredicted responses to global climate change, and that warming during a cool growing season enhances growth and alleviates stress.Field transcriptomics and metabolomics revealed that elevated temperature and [COThe online version of this article (10.1007/s11306-019-1511-8) contains supplementary material, which is available to authorized users. The International Panel on Climate Change (IPCC) has predicted that [CO2] will rise to 600 ppm and mean global temperature will increase 1.1 to 2.6 °C by the end of the century, with a moderate climate change projection scenario (IPCC 2014). Although the combined effects of rising atmospheric [CO2] and rising temperatures have been studied in field experiments in temperate forests  is a perennial C4 grass species native to tropical Africa, which has been adapted in Brazil as a forage and range grass  experiment was established in Ribeirão Preto, Brazil to expose Guinea grass to elevated [CO2] (600 ppm) and increased canopy temperatures of 2 °C  understand the biochemical responses of Guinea grass to rising [CO2] and temperature when applied individually and in combination; (2) understand how gene expression is altered in each treatment; and (3) integrate the metabolomic and transcriptional responses.Our study combines the power of FACE experiments for growing plants under elevated  plots (C), 4 elevated [CO2] (600 μmol mol−1) and ambient temperature plots (eC), 4 ambient [CO2] and elevated temperature [+ 2 °C] plots (eT), and 4 combined treatment (elevated [CO2] and elevated temperature) plots (eCeT). Each plot was 2-m in diameter; [CO2] was controlled using Free Air CO2 Enrichment, and canopy warming was provided by infrared ceramic heaters as described by Britto de Assis Prado et al. , and June 14, 2014 (time point B—50 days of treatment). Six leaves per experimental plot were collected at each time point and then pooled for RNA extraction and metabolomics analysis. At the time of sampling, tissue was immediately quenched in liquid nitrogen. RNA was extracted in Brazil using the protocol described by Bilgin and colleagues  were submitted for library construction and sequencing at the Roy J. Carver Biotechnology Center at the University of Illinois, Urbana-Champaign. RNAseq libraries were prepared with Illumina’s TruSeq Standard RNA sample prep kit according to the manufacturer’s instruction . No public reference genome for Guinea grass was available at the time of the sequencing. Therefore, a combination of MiSeq and HiSeq was used to generate sequences of different lengths for de novo genome assembly.RNA samples that were shipped from Brazil were re-suspended by washing with sodium acetate (pH 5.5) and glycerol. RNA quantity and quality were re-assessed using a spectrophotometer  and microfluidic visualization tool . 32 samples (16 per time point including 4 from each  on C4 metabolism , yet only a few genes were significantly differentially expressed in eT alone. Optimal growth for P. maximum is estimated to be at least 21 °C, and background temperature in ambient conditions during this experiment was only 17.0 ± 0.06 °C, so the eT treatment moved the plants closer to optimal growth temperatures   and general defense response to bacteria, suggesting greater stress in the eCeT treatment compared to control. The enrichment of SA biosynthesis transcripts has previously been identified to be part of the heat-stress response in plants. Salicylic acid stabilizes heat shock transcription factors increasing their binding to heat shock elements and thus promoting heat shock-related genes  from the WGCNA analysis contained significant numbers of DEG . The DEG identified within module 3 were enriched in salicylic acid (SA) biosynthesis . Switchgrass and maize mutants have shown that downregulation of COMT impacts lignin content of biofuel crops specifically decreasing the S-lignin units and improving ethanol yields  allowed us to test the correlation of leaf biomass and area with metabolite and transcript levels; however, no significant correlations were observed. The low temperatures reported on the day of sampling may have greatly affected the metabolite and transcript levels, hindering the correlation of these datasets with leaf measurements taken at the end of the season. Field conditions are also inherently more variable than controlled environment conditions, but field experiments better represent the variation expected in the future. After 50 d of treatment, we did observe significant correlations between transcripts and metabolites, with the most notable being xylose Table . Xylose Z. mays cinnamoyl-CoA reductase (CCR1). CCR1 is the entry point into the lignin branch of the phenylpropanoid pathway Supplementary material 2 (CSV 57 kb)Supplementary material 3 (CSV 8 kb)Supplementary material 4 (DOCX 967 kb)Below is the link to the electronic supplementary material.


We have identified two errors in text symbols:Figure 1 (C), the unit of light intensity used for optogenetic stimulation is incorrect: All instances of “mW/mm2” in this legend should be changed to “μW/mm2”. Correct text will be as follows:(1) In the legend of MZ699-Gal4 neurons with ~64 μW/mm2 200 ms whole-field 590 nm light elicited large depolarizations well above the spiking threshold in a MZ699 vPN (left); the average (black) of 10 trials (gray) from an example MZ699-vPN. The spikes the recorded neuron produced were small and difficult to see in the raw traces. A raw voltage trace of the same neuron upon stimulation with ~1.3 μW/mm2 light for 200 ms is shown in the inset. A stronger light stimulus (~64 μW/mm2 200 ms whole-field 590 nm light) delivered to the brain elicited large and reliable excitatory postsynaptic potentials (EPSPs) in the recorded ePNs (right); the average (black) of 10 trials (gray) from an example ePN in VM5v glomerulus is shown.Optogenetic activation of (2) In the subsection “Whole-Cell Patch Clamp Recordings” of section “MATERIALS AND METHODS”, in two places “≦2 day” should be changed to “≧2 day”. Correct text will be as follows:ex vivo recordings, brains of ≧2 day old flies were excised from the head capsule in extracellular saline and the perineural sheath above the ePN somata was removed with fine forceps. For in vivo recordings, the dorsal side of ≧2 day old female flies was restrained with gel epoxy on a plastic film with a small window over the fly's head.For The authors apologize for the mistakes. These errors do not change the scientific conclusions of the article in any way.The original article has been updated.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.


Deer antler is the only mammalian organ that can be completely regenerated every year. Its periodic regeneration is regulated by multiple factors, including transforming growth factor β (TGF-β). This widely distributed multi-functional growth factor can control the proliferation and differentiation of many types of cell, and it may play a crucial regulatory role in antler regeneration. This study explored the role of TGF-β1 during the rapid growth of sika deer antler.Three CRISPR-Cas9 knockout vectors targeting the TGF-β1 gene of sika deer were constructed and packaged with a lentiviral system. The expression level of TGF-β1 protein in the knockout cell line was determined using western blot, the proliferation and migration of cartilage cells in vitro were respectively determined using EdU and the cell scratch test, and the expression levels of TGF-β pathway-related genes were determined using a PCR array.Of the three gRNAs designed, pBOBI-gRNA2 had the best knockout effect. Knockout of TGF-β1 gene inhibits the proliferation of cartilage cells and enhances their migration in vitro. TGF-β signaling pathway-related genes undergo significant changes, so we speculate that when the TGF-β pathway is blocked, the BMP signaling pathway mediated by BMP4 may play a key role.TGF-β1 is a newly identified regulatory factor of rapid growth in sika deer antler. Deer antler, which is a secondary male sexual characteristic in most species of deer, is the only mammalian organ that can completely regenerate each year , 2. AntlThe development and regeneration of deer antler are associated with a variety of small biomolecules, including insulin-like growth factor (IGF), bone morphogenetic protein (BMP), epidermal growth factor (EGF), nerve growth factor (NGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and transforming growth factor (TGF) , 7. ThesTGF-β is a multi-functional cytokine that regulates cell proliferation, cell differentiation and extracellular matrix production. It is of great significance in development, wound healing, organ fibrosis and tumor metastasis . TGF-β hClustered regularly interspaced short palindromic repeats (CRISPRs) are a form of acquired immune system induced by RNA. The CRISPR immune defense mechanism evolved from bacteria and archaea, where it defends against the continuous attack of viruses and plasmids –14.The CRISPR system recognizes DNA through base pairing based on a pair of RNAs. It directs CRISPR-associated nuclease 9 (Cas9) to cleave the double-stranded DNA that it recognizes, resulting in a double-strand break (DSB) . The forCas9 is a naturally-occurring endonuclease with two enzyme cleavage domains: the HNH nuclease domain and the Ruv-C-like domain. They respectively cleave the complementary and non-complementary strands. In the cutting process, two assistant RNAs are needed for guidance in bacteria: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) , 19. TheOur previous immunohistochemistry results have confirmed that TGF-β1 was expressed in the skin, mesenchymal and cartilage layers of sika deer antler. The highest relative expression level was in the cartilage layer. In this study, we used CRISPR-Cas9 to knock out the TGF-β1 gene of antler cartilage cells, allowing the investigation of the effect of TGF-β1 on the growth and regeneration of sika deer antler.Sus scrofa, NM_214015.2), goat  and cattle  with high homology were obtained via blast alignment. The alignment of the three sequences showed that the positions of the exons and introns are substantially the same and the exons of sika deer TGF-β1 were predicted by referring to the three sequences.Since the whole genome sequence of sika deer has not been released, near-source species were used for gRNA design. A partial sequence of sika deer TGF-β1 was cloned via RT-PCR. The TGF-β1 sequences of pig  and the plasmids were extracted using an Endotoxin-free plasmid kit. Then, the lentiviruses were produced via packaging in 293 T cells.The three pairs of annealed gRNAs were ligated to the pBOBI vector. The recombinant plasmids that were positive for sequencing were named pBOBI-gRNA1, pBOBI-gRNA2 and pBOBI-gRNA3. After that, three plasmids  required for lentiviral packaging were transfected into 2 into tube B. Then we added the solution in tube B dropwise to tube A and incubated it for 10 min at room temperature. Finally, the transfection complex was slowly and uniformly added into 293 T cells and incubated at 37 °C for 12 h. The medium was changed for Dulbecco’s modified Eagle medium (DMEM) after 12 h, and virus supernatants were collected twice at 48 and 72 h.When the cells reached a growth density of 80%, we proceeded with calcium phosphate transfection. First, we prepared a lentiviral packaging plasmid mixture in the ratio pMDL:VSV-G:REV = 5:3:2, with a total concentration of 1 μg/μL. Second, we took two clean EP tubes (designated A and B). We added 400 μL 2 × HBS, 5 μg recombinant plasmid and 5 μg packing plasmid mixture into tube A and mixed. Simultaneously, we added an equal volume 2 × CaClCervus nippon) provided by the Jilin Agricultural University Deer Farm in Changchun, China. Cartilage tissues were isolated under a dissecting microscope. The tissues were digested with collagenase-I and hyaluronidase for 1.5 h at 37 °C, after which they were digested with collagenase-II for 3 h under the same conditions. After centrifugation, the cartilage cells were cultured in DMEM supplemented with 20% (v/v) fetal bovine serum (FBS), 200 U/mL penicillin and 100 U/mL streptomycin at 37 °C with 5% (v/v) CO2. This study was approved by the Ethical Committee for Laboratory Animals at Jilin Agricultural University (permit no. ECLA-JLAU-17031).Deer antler samples were obtained from a three-year old male sika deer , and observed using fluorescence microscopy to obtain the optimal MOI of antler cartilage cells.Cartilage cells were seeded into 12-well plates at a density of 10Next, the cells of each experimental group were infected with the optimal MOI, the polybrene was added at a final concentration of 8 μg/mL to increase the infection efficiency. After infection for 24 h, we changed the medium containing lentivirus for fresh complete medium and continued the culture at 37 °C. The medium was changed to G418-containing medium after 48 h. An equal concentration of G418 was added to wild-type cells as the control. We changed the medium every 2–3 days until the control cells were all dead. The stable knockout cell lines were thus obtained.The total proteins of the knockout cell lines were extracted, and the concentration of the collected proteins was determined using the protein assay kit via the BCA method. The relative expression level of TGF-β1 protein in the three knockout cell lines was detected via western blot to determine the gene knockout effect. After mixing with loading buffer, the protein was separated using 12% SDS-PAGE and transferred to PVDF membranes. Non-specific binding was blocked with 5% evaporated milk, and then the membranes were incubated with primary antibodies of TGF-β1 and GAPDH (Bioss) at 4 °C overnight. After that, the membranes were incubated with the secondary antibodies at room temperature for 2 h. After washing with TBST three times, the signals were detected using enhanced chemiluminescence (ECL) reagents and the protein band intensities were analyzed using Image-Pro Plus software. In this experiment, untreated cells were used as the normal control group, GFP-infected cells were used as the negative control group, and GAPDH was used as the internal reference.Cartilage cells were seeded into 96-well plates at a density of 4000 cells per well. We added 200 μL DMEM to each well for overnight growth. The cell proliferation of untreated group, negative control group and experimental group were examined after 24, 48 and 72 h. Cartilage cells were stained with EdU and Hoechst 33342 and cell proliferation was then evaluated using fluorescence microscope.First, we drew 3 uniform horizontal lines on the back of 12-well plates. Then, the transfected cells were seeded in the plates, giving full coverage, and incubated overnight. The next day, we used a pipette tip to draw three straight lines in vertical to the lines on the backs of the plates. Separated cells were washed away with PBS and cultured in serum-free medium for 24 h. We took pictures at 0, 6, 12 and 24 h.Total RNA was extracted from the knockout cell lines using the Trizol method. RNA purity and concentration were determined using the ultraviolet absorption method. The integrity of the RNA band was detected via capillary electrophoresis.2 SYBR Green Mastermix tube were centrifuged to the bottom of the tube and the PCR reaction mixture was prepared: 1350 μL of 2 × RT2 SYBR Green Mastermix, 102 μL of cDNA solution and 1248 μL of RNase-free water. Then, we added 25 μL of PCR reaction mixture into each well of RT2 Profiler PCR Array for real-time quantitative PCR. The procedure was 40 cycles of: 95 °C for 1 min, 95 °C for 15 s and 60 °C for 1 min. The expression levels of the TGF-β pathway-related genes in the control and experimental group were analyzed with heat maps and scatter plots.First, the total RNA was reverse transcribed to cDNA. The reagents in the RTp < 0.05 was considered statistically significant.All the experimental data are presented as means ± standard deviation (S.D.). SPSS 22.0 software was used for all the statistical analysis. The significance of differences between groups was evaluated using Student’s t test. We divided the exons of TGF-β1 and designed three gRNAs at the first exon (Fig. 8 TU/mL.The 293 T cells were infected with the collected lentivirus, and the expression of GFP protein after 48 h was observed via fluorescence microscopy Fig. b and c. The relative expression level of TGF-β1 protein was determined via western blot Fig. , and theThe cartilage cells were infected with pBOBI-gRNA2 lentivirus, and the proliferation of each experimental group was detected using the EdU method after culture for 24, 48 and 72 h. The proliferation of knockout cell was significantly lower than for the control group, and cartilage cell proliferation remained inhibited over time, indicating that the knockout of TGF-β1 can affect the proliferation of cartilage cells in vitro Fig. .Fig. 3EdThe changes in cartilage cell migration in the untreated, negative control and experimental groups were observed at 0, 6, 12 and 24 h. The results show that the cell migration in the experimental group was significantly higher than that in the other two groups, and no significant difference between the untreated and negative control groups appeared over time (Fig. In our PCR array experiment, untreated cartilage cells were used as the control, and TGF-β1 knockout cell lines were used as experimental groups. The heat map Fig. a and scaDeer antler has a stable growth cycle, grows with a fixed shape and position, and can be completely regenerated every year, making it an ideal mammalian morphogenesis model . PreviouActivation of AP and PP cells is primarily regulated by androgens, which stimulate them to produce a large quantity of cell growth factors that stimulate rapid antler growth . Among tIt is worth mentioning that the regulation of cell proliferation by TGF-β is particularly complicated. TGF-β may have different effects in different cell types. Also, for the same type of cells, TGF-β has a bidirectional regulation effect due to different stimulation doses .To further study the effect of TGF-β1 on antler regeneration and proliferation, we constructed three CRISPR-Cas9 knockout vectors targeting the TGF-β1 gene of sika deer. They were packaged using a lentiviral system, and we used lentivirus infection and G418 resistance screening to obtain a TGF-β1 knockout cell line of cartilage cells. The expression level of TGF-β1 protein in the knockout cell line was detected using western blot. pBOBI-gRNA2 showed the strongest and most stable knockout of TGF-β1, which indicated the second knockout site we designed is suitable. Then, we examined the effect of TGF-β1 on the proliferation and migration of cartilage cells in vitro. The results show that the lack of TGF-β1 inhibited the proliferation of cartilage cells in vitro, but promoted the migration of cartilage cells, which is of great significance to cartilage cells in sika deer.The influence of TGF-β1 on the expression level of TGF-β signaling pathway-related genes was detected using a PCR array. It showed that the knockout of the TGF-β1 gene resulted in both the upregulation and the downregulation of various related genes in the pathway. Further analysis revealed that the knockout of TGF-β1 mainly resulted in the upregulation of BMP4 and ID2 and the downregulation of BMPR2 and Smad1. Of these, ID2 is one of the most important target genes regulated by BMPs. It can be used as a negative or positive regulator of cell differentiation. BMPR2 and Smad1 are downstream molecules of BMP signaling. We speculate that when the TGF-β pathway is blocked, the BMP signaling pathway mediated by BMP4 may play a key role, and the specific mechanism needs further verification and exploration.Our research shows that TGF-β1 is a crucial regulatory factor in antler cartilage cells. It causes a decrease in cell proliferation and an increase in migration. It also activates the transmission of the BMP4 signaling pathway. However, the specific mechanism that mediates antler regeneration requires further study.