ActVI-ORFA directs metabolic flux towards actinorhodin by preventing intermediate degradation.

The biosynthetic pathway of actinorhodin in Streptomyces coelicolor A3(2) has been studied for decades as a model system of type II polyketide biosynthesis. The actinorhodin biosynthetic gene cluster includes a gene, actVI-orfA, that encodes a protein that belongs to the nuclear transport factor-2-like (NTF-2-like) superfamily. The function of this ActVI-ORFA protein has been a long-standing question in this field. Several hypothetical functions, including pyran ring cyclase, enzyme complex stability enhancer, and gene transcription regulator, have been proposed for ActVI-ORFA in previous studies. However, although the recent structural analysis of ActVI-ORFA revealed a solvent-accessible cavity, the protein displayed structural differences to the well-characterized cyclase SnoaL and did not possess a DNA-binding domain. The obtained crystal structure facilitates an inspection of the previous hypotheses regarding the function of ActVI-ORFA. In the present study, we investigated the effects of a series of actVI-orfA test plasmids with different mutations in an established vector/host system. Time-course analysis of dynamic metabolism profiles demonstrated that ActVI-ORFA prevented formation of shunt metabolites and may have a metabolic flux directing function, which shepherds the flux of unstable intermediates towards actinorhodin. The expression studies resulted in the isolation and structure elucidation of two new shunt metabolites from the actinorhodin pathway. Next, we utilized computational modeling to probe the active site of ActVI-ORFA and confirmed the importance of residues R76 and H78 in the flux directing functionality by expression studies. This is the first time such a function has been observed for a member of NTF-2-like superfamily in Streptomyces secondary metabolism.

Diverse Combinatorial Biosynthesis Strategies for C-H Functionalization of Anthracyclinones.

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Photoelectron Spectroscopy Reveals the Impact of Solvent Additives on Poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) Thin Film Formation.

The conductive polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is used in a manifold of electronic applications, and controlling its conductivity is often the key to attain a superior device performance. To that end, solvent additives like Triton, ethylene glycol (EG), or dimethyl sulfoxide (DMSO) are regularly incorporated. In our comprehensive study, we prepare PEDOT:PSS thin films with seven different additive combinations and with thicknesses ranging from 6 to 300 nm on indium-tin-oxide (ITO) substrates. We utilize X-ray photoelectron spectroscopy (XPS) to access the PSS-to-PEDOT ratio and the PSS--to-PSSH ratio in the near-surface region and ultraviolet photoelectron spectroscopy (UPS) to get the work function (WF). In addition, the morphology and conductivity of these samples are obtained. We found that the WF of the prepared thin films for each combination becomes saturated at a thickness of around 50 nm and thinner films show a lower WF due to the inferior coverage on the ITO. Furthermore, the WF shows a better correlation with the PSS--to-PSSH ratio than the commonly used PSS-to-PEDOT ratio as PSS- can directly affect the surface dipole. By adding solvent additives, a dramatic increase in the conductivity is observed for all PEDOT:PSS films, especially when DMSO is involved. Moreover, adding the additive Triton (surfactant) helps to suppress the WF fluctuation for most films of each additive combination and contributes to weaken the surface dipole, eventually leading to a lower and thickness-independent WF.

Tuning the Surface Electron Accumulation Layer of In2 O3 by Adsorption of Molecular Electron Donors and Acceptors.

In2 O3 , an n-type semiconducting transparent transition metal oxide, possesses a surface electron accumulation layer (SEAL) resulting from downward surface band bending due to the presence of ubiquitous oxygen vacancies. Upon annealing In2 O3 in ultrahigh vacuum or in the presence of oxygen, the SEAL can be enhanced or depleted, as governed by the resulting density of oxygen vacancies at the surface. In this work, an alternative route to tune the SEAL by adsorption of strong molecular electron donors (specifically here ruthenium pentamethylcyclopentadienyl mesitylene dimer, [RuCp*mes]2 ) and acceptors (here 2,2'-(1,3,4,5,7,8-hexafluoro-2,6-naphthalene-diylidene)bis-propanedinitrile, F6 TCNNQ) is demonstrated. Starting from an electron-depleted In2 O3 surface after annealing in oxygen, the deposition of [RuCp*mes]2 restores the accumulation layer as a result of electron transfer from the donor molecules to In2 O3 , as evidenced by the observation of (partially) filled conduction sub-bands near the Fermi level via angle-resolved photoemission spectroscopy, indicating the formation of a 2D electron gas due to the SEAL. In contrast, when F6 TCNNQ is deposited on a surface annealed without oxygen, the electron accumulation layer vanishes and an upward band bending is generated at the In2 O3 surface due to electron depletion by the acceptor molecules. Hence, further opportunities to expand the application of In2 O3 in electronic devices are revealed.

A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor.

Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-ß-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15-20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-ß-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.

Plastomes of Bletilla (Orchidaceae) and Phylogenetic Implications.

The genus Bletilla is a small genus of only five species distributed across Asia, including B. chartacea, B. foliosa, B. formosana, B. ochracea and B. striata, which is of great medicinal importance. Furthermore, this genus is a member of the key tribe Arethuseae (Orchidaceae), harboring an extremely complicated taxonomic history. Recently, the monophyletic status of Bletilla has been challenged, and the phylogenetic relationships within this genus are still unclear. The plastome, which is rich in both sequence and structural variation, has emerged as a powerful tool for understanding plant evolution. Along with four new plastomes, this work is committed to exploring plastomic markers to elucidate the phylogeny of Bletilla. Our results reveal considerable plastomic differences between B. sinensis and the other three taxa in many aspects. Most importantly, the specific features of the IR junction patterns, novel pttRNA structures and codon aversion motifs can serve as useful molecular markers for Bletilla phylogeny. Moreover, based on maximum likelihood and Bayesian inference methods, our phylogenetic analyses based on two datasets of Arethuseae strongly imply that Bletilla is non-monophyletic. Accordingly, our findings from this study provide novel potential markers for species identification, and shed light on the evolution of Bletilla and Arethuseae.

Optimization of Ultrasound-Assisted Extraction of Polyphenols from Ilex latifolia Using Response Surface Methodology and Evaluation of Their Antioxidant Activity.

The polyphenolic extract of Ilex latifolia (PEIL) exhibits a variety of biological activities. An evaluation of the parameters influencing the ultrasonic extraction process and the assessment of PEIL antioxidant activity are presented herein. Response surface methodology (RSM) was used to optimize the experimental conditions for the polyphenols ultrasonic-assisted extraction (UAE) from the leaves of Ilex latifolia. We identified the following optimal conditions of PEIL: ethanol concentration of 53%, extraction temperature of 60 °C, extraction time of 26 min and liquid−solid ratio of 60 mL/g. Using these parameters, the UAE had a yield of 35.77 ± 0.26 mg GAE/g, similar to the value we predicted using RSM (35.864 mg GAE/g). The antioxidant activity of PEIL was assessed in vitro, using various assays, as well as in vivo. We tested the effects of various doses of PEIL on D-galactose induced aging. Vitamin C (Vc) was used as positive control. After 21 days of administration, we measured superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, malondialdehyde (MDA) levels in mouse serum and liver tissue. The results demonstrated that the PEIL exhibits potent radical scavenging activity against 1,1-diphenyl-2-picrythydrazyl (DPPH∙), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+), and hydroxyl (∙OH) radicals. The serum concentrations of SOD and GSH-Px were higher, and MDA levels were lower, in the medium- and high-dose PEIL-treated groups than those in the aging group (p < 0.01), and the activity of MDA was lower than those of the model group (p < 0.01). The liver concentrations of SOD and GSH-Px were higher (p < 0.05), and MDA levels were lower, in the medium- and high-dose PEIL-treated groups than those in the aging control group (p < 0.01). These results suggest that optimizing the conditions of UAE using RSM could significantly increase the yield of PEIL extraction. PEIL possesses strong antioxidant activity and use as a medicine or functional food could be further investigated.

Hypersecretion of OmlA antigen in Corynebacterium glutamicum through high-throughput based development process.

Outer membrane lipoprotein A (OmlA) is a vaccine antigen against porcine contagious pleuropneumonia (PCP), a disease severely affecting the swine industry. Here, we aimed to systematically potentiate the secretory production of OmlA in Corynebacterium glutamicum (C. glutamicum), a widely used microorganism in the food industry, by establishing a holistic development process based on our high-throughput culture platform. The expression patterns, expression element combinations, medium composition, and induction conditions were comprehensively screened or optimized in microwell plates (MWPs), followed by fermentation parameter optimization in a 4 × 1 L parallel fermentation system (CUBER4). An unprecedented yield of 1.01 g/L OmlA was ultimately achieved in a 5-L bioreactor following the scaling-up strategy of fixed oxygen mass transfer coefficient (kLa), and the produced OmlA antigen showed well-protective immunity against Actinobacillus pleuropneumoniae challenge. This result provides a rapid and reliable pipeline to achieve the hyper-production of OmlA, and possibly other recombinant vaccines, in C. glutamicum. KEY POINTS: • Established a holistic development process and applied it to potentiate the secretion of OmlA. • The secretion of OmlA reached an unprecedented yield of 1.01 g/L. • The recombinant OmlA antigen induced efficient protective immunity.

Study of the genus Torreya (Taxaceae) based on chloroplast genomes.

BACKGROUND: Species of the genus Torreya are similar in morphology, and their morphological taxonomic characteristics are not stable because of environmentally induced changes. Therefore, morphology is insufficient for understanding their relationships. Chloroplast genome sequencing technology provides a powerful tool for molecular analysis to get more infomation for classification and identification of Torreya genus. METHODS: A total of 4 chloroplast genome of Torreya, including T. Parvifolia, T. nucifera, T. fargesii var. Yunnanensis and T. grandis var. jiulongshanensis, were sequenced and annotated. Campartive genome and phylogenetic tree were provided for variation analysis. RESULTS: The chloroplast genome size of the four samples is about 137 kb, the inverted repeat (IR) regions are identified in the genus Torreya. Genome comparison using mVISTA showed high sequence similarity among different species. Regions with divergence in exon regions include accD, ndhB, ndhF, psbA, psbJ, rpl2, rps3, rps16, rps18, ycf1, and ycf2. The phylogenetic tree based on 73 single-copy genes showed a clearer relationships among different species of Torreya. CONCLUSIONS: All genomes of the four Torreya species consist of two short IR regions, and results of the phylogenetic analysis concluded that T. parvifolia should be considered as T. fargesii var. yunnanensis or treated as a sister species. T. grandis var. jiulongshanensis should be treated as a variety of T. grandis according to molecular evidence, supporting the originally published proposal.

Comprehensive evaluation of Bletilla striata and its substitutes by combining phenotypic characteristic, chemical composition, and anti-melanogenic activity.

Bletilla striata is a precious traditional Chinese medical herb with a wide range of applications in pharmacological and cosmetic fields. Because of the shortage of resources, Bletilla ochracea and Bletilla formosanare are also used as the substitutes. To distinguish the differences and homologies, the typical morphologic and microscopic characteristics of them were compared, and a UPLC fingerprint analysis coupled with chemometric methods were developed for characterization and quality evaluation of Bletillae Rhizoma. Gastrodin, protocatechuic acid, gymnoside V, blestrianol A, coelonin, gymnosides Ⅸ and batatasin II were identified as the potential chemical markers for comprehensive quality evaluation by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The anti-melanogenic activities of the three species were also compared for the first time in vivo using the zebrafish model, the results suggested that B. striata and its two substitutes had obvious anti-melanogenic activities, and they were not-toxic at depigmenting doses. Molecular docking studies revealed batatasin III, blestrianol A, coelonin, and gastrodin were possible multitarget compounds associated with melanogenesis suppression, which are important for their potential future medical application.

A Link Between Chemical Structure and Biological Activity in Triterpenoids.

BACKGROUND: Plants that are rich in triterpenoid compounds possess various biological activities and are reported in many scientific works. Triterpenoids, because of their broad sources, wide variety, high medicinal value, and anti-tumor properties, have drawn great attention from scientists. However, the lack of approach to understand the link between their chemical structures and biological activities has limited the fundamental comprehension of these compounds in cancer therapy. OBJECTIVE: The aim of the study is to summarize the list of plants with triterpenoids and their derivatives that are a source of potential novel therapeutic anti-cancer agents by interpreting the network of anti-cancer activity and the structures of triterpenoids and their derivatives. METHODS: This work focuses on analyzing relevant patents and references that detail the structure of triterpenoids and their derivatives for the treatment of tumors. RESULTS: Pentacyclic triterpenoid plays a more important role in improving the autophagic signaling pathways of cancer cells compared to tetracyclic triterpenoid. CONCLUSION: The heterogenous skeleton structure of triterpenoids impairs programmed cell death signaling pathways in various cancers.

Secondary Phosphine Oxide Functionalized Gold Clusters and Their Application in Photoelectrocatalytic Hydrogenation Reactions.

Ligands in ligand-protected metal clusters play a crucial role, not only because of their interaction with the metal core, but also because of the functionality they provide to the cluster. Here, we report the utilization of secondary phosphine oxide (SPO), as a new family of functional ligands, for the preparation of an undecagold cluster Au11-SPO. Different from the commonly used phosphine ligand (i.e., triphenylphosphine, TPP), the SPOs in Au11-SPO work as electron-withdrawing anionic ligands. While coordinating to gold via the phosphorus atom, the SPO ligand keeps its O atom available to act as a nucleophile. Upon photoexcitation, the clusters are found to inject holes into p-type semiconductors (here, bismuth oxide is used as a model), sensitizing the p-type semiconductor in a different way compared to the photosensitization of a n-type semiconductor. Furthermore, the Au11-SPO/Bi2O3 photocathode exhibits a much higher activity toward the hydrogenation of benzaldehyde than a TPP-protected Au11-sensitized Bi2O3 photocathode. Control experiments and density functional theory studies point to the crucial role of the cooperation between gold and the SPO ligands on the selectivity toward the hydrogenation of the C═O group in benzaldehyde.

Atranones from Stachybotrys chartarum and their antitumor activities in MG-63 human osteosarcoma cells.

Two new atranones T and U (1 and 2), and three known analogues atranone B (3), atranone Q (4), and stachatranone C (5) were isolated from the toxigenic fungus Stachybotrys chartarum. Their structures and absolute configurations were elucidated by spectroscopic data and calculated ECD analyses. The cytotoxicities of all the atranones (1-5) were evaluated against MG-63 human osteosarcoma cell lines. Compound 4 exhibited significant cytotoxic effect against MG-63 with IC50 value of 8.6 µM, being more active than the positive control, 5-FU (IC50 10.4 µM). Morphological features of apoptosis activities were evaluated in 4-treated MG-63 cells. Compound 4 effectively induced apoptosis of MG-63, which was associated with G0/G1-phase cell cycle arrest. Flow cytometric analysis showed that the treatment by 4 significantly induced MG-63 cell apoptosis in a dose-dependent manner.

Differentiation between wild and artificial cultivated Stephaniae tetrandrae radix using chromatographic and flow-injection mass spectrometric fingerprints with the aid of principal component analysis.

High-performance liquid chromatographic (HPLC) and flow-injection mass spectrometric (FIMS) fingerprinting profiles were used to differentiate between wild and artificial cultivated Stephaniae tetrandrae Radix samples. HPLC and FIMS fingerprints of 15 wild S. tetrandrae Radix samples and 12 artificial cultivated S. tetrandrae Radix samples were obtained and analyzed with the aid of principal component analysis (PCA). PCA of the fingerprints showed that the chemical differences between wild and artificial cultivated S. tetrandrae Radix samples could be differentiated by either HPLC or FIMS fingerprints. The HPLC fingerprints provided more chemical information but required longer analytical time compared with FIMS fingerprints. This study indicated that the wild samples contained higher concentrations of almost all of the major compounds than the cultivated samples. Three characteristic compounds which were responsible for the differences between the samples were tentatively identified with the aid of MS data. Furthermore, these three compounds, tetrandrine (TET), fangchinoline (FAN), and cyclanoline (CYC), were quantified. The HPLC and FIMS fingerprints combined with PCA could be used for quality assessment of wild and artificial cultivated S. tetrandrae Radix samples.

A scale-down model of 4000-L cell culture process for inactivated foot-and-mouth disease vaccine production.

The anticipated increasing demand for inactivated foot-and-mouth (FMD) disease vaccine calls for its larger production capacity, while development of a large-scale process typically requires high running cost and has very limited experimental throughput at manufacturing scale. Thus, an economic scale-down model of representing a large-scale process becomes necessary and essential. In this study, we used a systematic approach to establish a scale-down model representing a 4000-L culture process for FMD vaccine production by suspension BHK-21 cells. In detail, we firstly compared hydrodynamic properties of three bioreactors (14-L, 800-L and 4000-L) under three different conditions (equivalent mixing time, equivalent shear stress and equivalent volumetric power). We figured out equivalent volumetric power (P/V) potentially as an appropriate scale-down strategy, since it resulted in comparable calculated hydrodynamic parameters among three bioreactors. Next, we used computational fluid dynamics (CFD) simulation to provide more details about hydrodynamic environments inside the bioreactors, which supports the reliability of this scale-down strategy. Finally, we compared cell growth, metabolites, vaccine productivity and product quality attributes during FMD vaccine production by BHK-21 cells and observed very close performances among three bioreactors, which once again demonstrates the robustness of this scale-down model. This scale-down strategy can be applied to study variations and critical quality attributes (CQAs) in the resultant production process based on quality by design (QbD) principles, aiming at further more efficient optimization of vaccine production.

Quality by Design-Driven Process Development of Severe Fever With Thrombocytopenia Syndrome Vaccine.

Owing to the biological activity of the vaccine, the complicated production process, sterility, and uniformity of the product, the producing process of the vaccine is complicated and the product quality hard to control. In recent years, with the development of basic science such as cell biology, molecular biology, and metabolic engineering, bioprocess engineering research has developed rapidly. Therefore, U.S. Food and Drug Administration and European Medicines Agency conduct stringent control over the development of biomedical process engineering and product quality. This case study describes an example of Quality by Design-driven process development for manufacturing a human vaccine produced with Vero cells. Cell density in harvest fermentation broth and antigenic titer were chosen as 2 critical quality attributes. The study through 3 rounds design of experiment revealed that H2O2 and cell boost 4 had a significant effect on antigenic titer. Ethanolamine had significant improvement in the final concentration of cells. Through the Monte Carlo simulation, the design spaces and control space of process parameters were determined. A successful validation in a bioreactor was executed to verify the results of a spinner flask. Our investigation presents a successful case of Quality by Design principle, which encourages other researchers to combine the methodology into other biopharmaceutical manufacturing process.

Clematisguniuensis (Ranunculaceae), a new species from Eastern China.

Clematisguniuensis sp. nov., a new narrowly endemic species of Clematis, is described and illustrated from the Huangshan Mountains of Eastern China. A description of C.guniuensis is presented along with illustrations, photographs and diagnostic differences between the new species and its putative close allies.

Discovery of an EGFR tyrosine kinase inhibitor from Ilex latifolia in breast cancer therapy.

Deficient signaling of the EGFR and other receptor tyrosine kinases in humans is associated with diseases such as cancer. Some EGFR tyrosine kinase inhibitors have become a new class of targeted therapeutic agents in the last years. We found that 27-O-p-(E)-coumaroyl ursolic acid (27-CAUA) had a strong activity of apoptosis according to preparation by screening for a series of Ilex latifolia products. 27-CAUA inhibited EGFR kinase system to lead to inactivation of PI3K/AKT/mTOR and Ras-Raf-MEK-ERK signal pathways which implicated in the proliferation and survival of tumor cells. These findings suggested that 27-CAUA was an orally active, selective epidermal growth factor receptor-tyrosine kinase inhibitor which could lead to beneficial manifestations in the clinic.

Quality by Design-Driven Process Development of Cell Culture in Bioreactor for the Production of Foot-And-Mouth Veterinary Vaccine.

Quality by design (QbD) principle has been established as a guideline to emphasize the understanding of the relationship of product quality with process control. Vaccine product have characteristics of security and high efficiency, but it also has features such as complexity and rigorous regulatory for production. This case study describes an example of QbD-driven process development for manufacturing a veterinary vaccine produced with baby hamster kidney-21 cells. The study revealed that cell culture duration was the most significant factor affecting 50% tissue culture infectious doses (TCID50) and antigenic titer, and the factors of culture temperature and pH at infection phase exhibited less effect. Culture temperature at infection phase was the only significant factor for total protein. Through the Monte Carlo simulation, the design spaces of process parameters were determined. Meanwhile, the excellent and robust performance in manufacturing scale (4000-L) validated the effectiveness of this strategy. A reliable and robust multivariate process parameter range, that is, design space, was identified by this systematic approach. Our investigation presents a successful case of QbD principle, which encourages other researchers to combine the methodology into other biopharmaceutical manufacturing process.

Constructing the Electronic Structure of CH3NH3PbI3 and CH3NH3PbBr3 Perovskite Thin Films from Single-Crystal Band Structure Measurements.

Photovoltaic cells based on halide perovskites, possessing remarkably high power conversion efficiencies have been reported. To push the development of such devices further, a comprehensive and reliable understanding of their electronic properties is essential but presently not available. To provide a solid foundation for understanding the electronic properties of polycrystalline thin films, we employ single-crystal band structure data from angle-resolved photoemission measurements. For two prototypical perovskites (CH3NH3PbBr3 and CH3NH3PbI3), we reveal the band dispersion in two high-symmetry directions and identify the global valence band maxima. With these benchmark data, we construct "standard" photoemission spectra from polycrystalline thin film samples and resolve challenges discussed in the literature for determining the valence band onset with high reliability. Within the framework laid out here, the consistency of relating the energy level alignment in perovskite-based photovoltaic and optoelectronic devices with their functional parameters is substantially enhanced.